Polyol oxidases

ABSTRACT

The present invention provides compositions and methods for producing a polyol oxidase in micoroorganisms, and the use of polyol oxidases in cleaning compositions. The invention includes cleaning compositions that contain combinations of two or more POx oxidases, and cleaning compositions that contain combinations of two or more POx oxidases and a perhydrolase. In particular, the invention provides methods for expressing polyol oxidases in bacterial hosts for use in detergent applications for cleaning, bleaching and disinfecting.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application U.S.60/853,227, filed Oct. 20, 2006; U.S. provisional application U.S.60/853,258, filed on Oct. 20, 2006; International PCT ApplicationDK2006/000590, filed on Oct. 20, 2006, which claims priority toProvisional Application 200501474, filed on Oct. 21, 2005; and toInternational PCT Application DK2006/000591, filed on Oct. 20, 2006,which claims priority to Danish Application PA200501474, filed on Oct.21, 2005, all of which are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for producing apolyol oxidase in micoroorganisms, and the use of polyol oxidases incleaning compositions. The invention includes cleaning compositions thatcontain combinations of two or more POx oxidases, and cleaningcompositions that contain combinations of two or more POx oxidases and aperhydrolase. In particular, the invention provides methods forexpressing polyol oxidases in bacterial hosts for use in detergentapplications for cleaning, bleaching and disinfecting.

BACKGROUND OF THE INVENTION

Oxidoreductases are enzymes that catalyze the transfer of electrons fromone molecule (the reductant, also called the hydrogen acceptor orelectron donor) to another (the oxidant, also called the hydrogen donoror electron acceptor). Oxidoreductases are classified as EC 1 in the ECnumber classification of enzymes. Oxidoreductases can be furtherclassified into 22 subclasses, and the oxidoreductases belonging to ECclass 1.1.3. act on the CH—OH group of donors with oxygen as acceptor.The oxidoreductase enzymes of the EC1.1.3 class of enzymes are oxidases,and their ability to generate hydrogen peroxide has found use inimproving the storage stability of food products including cheese,butter, meat, wine and fruit juice (See e.g., Hammer, Oxidoreductases:Enzymes in Food Processing, Nagodawithana and Reed (eds). AcademicPress, NY, [1998]; pp. 251-254; and Tiina and Sandholm, Int. J. FoodMicrobiol., 8:165-74 [1989]). Indeed, oxidases have found use ascomponents of various compositions, including foods, personal careitems, and detergents. However, there remains a need for methods andcompositions that facilitate the efficient and economical production ofoxidases for use in any suitable setting.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for producing apolyol oxidase in micoroorganisms, and the use of polyol oxidases incleaning compositions. The invention includes cleaning compositions thatcontain combinations of two or more POx oxidases, and cleaningcompositions that contain combinations of two or more POx oxidases and aperhydrolase. In particular, the invention provides methods forexpressing polyol oxidases in bacterial hosts for use in detergentapplications for cleaning, bleaching and disinfecting.

In one embodiment, the invention provides an isolated chimericpolynucleotide comprising a sequence encoding a mature polyol oxidaseprotein (POx) that is operably linked to a sequence encoding a secretorysignal peptide, which is derived from a prokaryotic microorganism.

In another embodiment, the invention provides an isolated chimericpolynucleotide that encodes a mature polyol oxidase protein (POx) thatis operably linked to a sequence encoding a secretory signal peptidederived from a Streptomyces sp., an Acidothermus sp. or an Arthrobactersp.

In another embodiment, the invention provides for an isolated chimericpolynucleotide that encodes a polypeptide selected form SEQ ID NO:16,28, 35, 37, 38, and 40, wherein the chimeric polynucleotide comprises asequence encoding a mature polyol oxidase protein (POx) operably linkedto a sequence encoding a secretory signal peptide, which is derived froma prokaryotic microorganism.

In another embodiment, the invention provides an isolated chimericpolynucleotide comprising a sequence encoding a mature polyol oxidaseprotein (POx) that is operably linked to a sequence encoding a secretorysignal peptide, which is peptide is selected from the signal sequenceencoded by the S. coelicolor gene SCO7637, the signal sequence encodingthe S. lividans gene SCO 0624 and the signal sequence encoding the B.subtilis gene P43379.

In another embodiment, the invention provides for a recombinantexpression vector comprising an isolated chimeric polynucleotide thatcomprises a sequence that encodes a mature polyol oxidase protein (POx)that is operably linked to a sequence encoding a secretory signalpeptide derived from a Streptomyces sp., an Acidothermus sp. or anArthrobacter sp.

In another embodiment, the invention provides a host cell that comprisesa recombinant expression vector that an comprises isolated chimericpolynucleotide that comprises a sequence that encodes a mature polyoloxidase protein (POx) that is operably linked to a sequence encoding asecretory signal peptide derived from a Streptomyces sp., anAcidothermus sp. or an Arthrobacter sp.

In another embodiment, the invention provides a host cell that comprisesa polynucleotide that encodes a POx polypeptide that has a sequenceselected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 28,35, 37, 38 and 40.

In another embodiment, the invention provides a host cell that comprisesa polynucleotide that encodes a POx polypeptide that has sorbitoloxidase and/or xylitol oxidase activity, and that has a sequenceselected from SEQ ID NO 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 28,35, 37, 38 and 40.

In another embodiment, the invention provides a host cell that comprisesa polynucleotide that polynucleotide is present in the genome of saidhost cell or in a vector that autonomously replicates in said host cell,and that encodes a POx polypeptide that has a sequence selected from SEQID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 28, 35, 37, 38 and40.

In another embodiment, the invention provides an S. lividans, a B.subtilis or an Acidothermus cellulolyticus host cell host cell thatcomprises a polynucleotide that encodes a POx polypeptide that has asequence selected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 16, 28, 35, 37, 38 and 40.

In another embodiment, the invention provides a method for producing apolypeptide having POx activity that includes (a) transforming a hostcell with a recombinant expression vector that comprises apolynucleotide sequence that encodes a POx polypeptide having a sequenceselected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,28, 35, 37, 38 and 40, (b) growing the transformed host cell underconditions suitable for the expression of said POx polypeptide; and (c)recovering the POx polypeptide.

In another embodiment, the invention provides a method for producing apolypeptide having sorbitol and/or xylitol activity that includes (a)transforming a host cell with a recombinant expression vector thatcomprises a polynucleotide sequence that encodes a POx polypeptidehaving a sequence selected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 16, 28, 35, 37, 38 and 40, (b) growing the transformed hostcell under conditions suitable for the expression of said POxpolypeptide; and (c) recovering the POx polypeptide.

In another embodiment, the invention provides a method for producing apolypeptide having POx activity that includes (a) transforming aBacillus, a Streptomyces or an E. Coli host cell with a recombinantexpression vector that comprises a polynucleotide sequence that encodesa POx polypeptide having a sequence selected from SEQ ID NO: 2, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 16, 28, 35, 37, 38 and 40, (b) growing thetransformed host cell under conditions suitable for the expression ofsaid POx polypeptide; and (c) recovering the POx polypeptide.

In another embodiment, the invention provides a method for producing apolypeptide having POx activity that includes (a) transforming a hostcell with a recombinant expression vector that comprises apolynucleotide sequence that encodes a POx polypeptide having a sequenceselected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,28, 35, 37, 38 and 40, (b) growing the transformed host cell underconditions suitable for the expression of said POx polypeptide; and (c)recovering the POx polypeptide, wherein the expression of thepolypeptide is extracellular.

In another embodiment, the invention provides a method for producing apolypeptide having POx activity that includes (a) transforming a hostcell with a recombinant expression vector that comprises apolynucleotide sequence that encodes a POx polypeptide having a sequenceselected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,28, 35, 37, 38 and 40, (b) growing the transformed host cell underconditions suitable for the expression of said POx polypeptide; and (c)recovering the POx polypeptide, wherein the expression of thepolypeptide is intracellular.

In another embodiment, the invention provides for a cleaning compositionthat comprises an effective amount of an isolated POx that has an aminoacid sequence that is at least about 70% identical to a POx having asequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and/or 14.

In another embodiment, the invention provides for a detergentcomposition that comprises an effective amount of an isolated POx thathas an amino acid sequence that is at least about 70% identical to a POxhaving a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, and/or 14.

In another embodiment, the invention provides for a detergentcomposition that comprises an effective amount of an isolated POx thathas an amino acid sequence that is at least about 70% identical to a POxhaving a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, and/or 14, at least one additional enzyme, and a bleachactivator.

In another embodiment, the invention provides for a cleaning compositionthat comprises an effective amount of an isolated POx that has an aminoacid sequence that is at least about 70% identical to a POx having asequence selected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and/or 14 and least one additional enzyme.

In another embodiment, the invention provides for a cleaning compositionthat comprises an effective amount of an isolated POx that has an aminoacid sequence that is at least about 70% identical to a POx having asequence selected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and/or 14 and least one additional enzyme that is selected fromhemicellulases, peroxidases, proteases, cellulases, xylanases, lipases,phospholipases, esterases, cutinases, pectinases, keratinases,reductases, oxidases, oxidoreductases, perhydrolases, phenoloxidases,lipoxygenases, ligninases, pullulanases, tannases, pentosanases,mannanases, β-glucanases, arabinosidases, hyaluronidases,chondroitinases, laccases, and amylasess, or mixtures thereof.

In another embodiment, the invention provides for a cleaning compositionthat comprises an effective amount of an isolated POx that has an aminoacid sequence that is at least about 70% identical to a POx having asequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and/or 14 and least one additional enzyme that is a perhydrolase.

In another embodiment, the invention provides for a cleaning compositionthat comprises an effective amount of an isolated POx that has an aminoacid sequence that is at least about 70% identical to a POx having asequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and/or 14 and least one additional enzyme that is a glucose oxidase.

In another embodiment, the invention provides for a bleachingcomposition that comprises an effective amount of an isolated POx thathas an amino acid sequence that is at least about 70% identical to a POxhaving a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, and/or 14 and least one additional enzyme that is a glucoseoxidase.

In another embodiment, the invention provides for a method of cleaningthat includes contacting a hard surface and/or an article comprising afabric with a cleaning composition that comprises an effective amount ofan isolated POx that has an amino acid sequence that is at least about70% identical to a POx having a sequence selected from SEQ ID NO:2, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, and/or 14.

In another embodiment, the invention provides for a method of cleaningthat includes contacting a hard surface and/or an article comprising afabric with a cleaning composition that comprises an effective amount ofan isolated POx that has an amino acid sequence that is at least about70% identical to a POx having a sequence selected from SEQ ID NO:2, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, and/or 14, and rinsing said surfaceand/or article after contacting said surface or article with saidcleaning composition.

In another embodiment, the invention provides for a method of cleaningthat includes contacting a hard surface and/or an article comprising afabric stained with a substance containing at least one polylol, with acleaning composition that comprises an effective amount of an isolatedPOx that has an amino acid sequence that is at least about 70% identicalto a POx having a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, and/or 14.

In another embodiment, the invention provides for a method of cleaningthat includes contacting a hard surface and/or an article comprising afabric stained with a substance containing at least one polylol selectedfrom the group of D-sorbitol, D-xylitol, D-mannitol, D-ribitol,myo-inositol, glycerol, 1,3,-propanediol and 1,2-propanediol, with acleaning composition that comprises an effective amount of an isolatedPOx that has an amino acid sequence that is at least about 70% identicalto a POx having a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, and/or 14.

In another embodiment, the invention provides for a method of cleaningthat includes contacting a hard surface and/or an article comprising afabric, which is soiled with juice, wine and/or tea, with a cleaningcomposition that comprises an effective amount of an isolated POx thathas an amino acid sequence that is at least about 70% identical to a POxhaving a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, and/or 14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a map of expression plasmid pKB105-TAT-50×7775 (SEQ IDNO:29).

FIG. 2 provides a map of expression plasmid pKB105-CelA-SOx-7775 (SEQ IDNO:30).

FIG. 3 provides a map of expression plasmid pet24a-SOx (neutral codons).

FIG. 4 provides a map of expression plasmid pSMM-SOx S. lividans.

FIG. 5 provides a graph showing the results of a washing study todetermine the ability of sorbitol oxidase to remove tea stains on cottondiscs.

FIG. 6 provides a graph showing the results of a SOx dose study forbleaching of tea stains on cotton discs.

FIG. 7 provides a graph showing the results of a washing and bleachingperformance study of sorbitol oxidase to remove of wine stains on cottondiscs.

FIG. 8 provides a graph showing the results of a bleaching performancestudy of the ability of sorbitol oxidase to remove blueberry stains oncotton discs.

FIG. 9 (A-B) show the rate of H2O2 production by a combination of aconstant amount of a first polyol oxidase (SOx) with an increasingamount of a second polyol oxidase (HOx or GOx). The amount of GOx or HOxrelative to the constant amount of SOx was increased between 1× and3000× (A) or 1× and 100× (B), and the rate of H2O2 production wasmeasured over 5 minutes in a 300 ul ABTS assay. The combination of SOxwith HOx (♦) or SOx with GOx (▪) resulted in a synergistic increase inthe production of H2O2 of between 250 and 300% of that seen with SOxalone ( - - - ) at 3000× (A).

DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods for producing apolyol oxidase in micoroorganisms, and the use of polyol oxidases incleaning compositions. The invention includes cleaning compositions thatcontain combinations of two or more POx oxidases, and cleaningcompositions that contain combinations of two or more POx oxidases and aperhydrolase. In particular, the invention provides methods forexpressing polyol oxidases in bacterial hosts for use in detergentapplications for cleaning, bleaching and disinfecting.

DEFINITIONS

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, recombinant DNA fields, and industrial enzyme use anddevelopment, all of which are within the skill of the art. All patents,patent applications, articles and publications mentioned herein, bothsupra and infra, are hereby expressly incorporated herein by referencein their entirety.

Furthermore, the headings provided herein are not limitations of thevarious aspects or embodiments of the invention, which can be had byreference to the specification as a whole. Accordingly, the termsdefined immediately below are more fully defined by reference to thespecification as a whole. Nonetheless, in order to facilitateunderstanding of the invention, definitions for a number of terms areprovided below.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For example,Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Margham,The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991)provide those of skill in the art with a general dictionaries of many ofthe terms used in the invention. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceof the present invention, preferred methods and materials are describedherein. Accordingly, the terms defined immediately below are more fullydescribed by reference to the Specification as a whole. Also, as usedherein, the singular terms “a,” “an,” and “the” include the pluralreference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary, depending upon the context theyare used by those of skill in the art.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

As used herein, the term “oxidase” refers to enzymes that catalyze anoxidation/reduction reaction involving molecular oxygen (O₂) as theelectron acceptor. In these reactions, oxygen is reduced to water (H₂O)or hydrogen peroxide (H₂O₂). The oxidases are a subclass of theoxidoreductases.

As used herein, “polyol oxidase”, “POx” or “POx polypeptide” refers toenzymes that catalyze the oxidation of various polyols (e.g., sorbitol,xylitol, arabitol, mannitol, ribitol, glycerol, propanediol, andpropylene glycol). Polyol oxidase enzymes include oxidoreductase enzymeof the EC1.1.3 class of enzymes that includes sorbitol oxidase, xylitoloxidase, glucose oxidase, hexose oxidase, alcohol oxidase, cholineoxidase, mannitol oxidase. As used herein, “polyol” refers to chemicalcompounds that contain multiple hydroxyl groups.

As used herein, the term “glucose oxidase” (“GOx”) refers to the oxidaseenzyme (EC 1.1.3.4), a dimeric protein which catalyzes the oxidation ofbeta-D-glucose into D-glucono-1,5-lactone, which then hydrolyzes togluconic acid with concomitant reduction of molecular oxygen to hydrogenperoxide.

As used herein, the term “alcohol oxidase” (“AOx”) refers to the oxidaseenzyme (EC 1.1.3.13) that converts an alcohol to an aldehyde withconcomitant reduction of molecular oxygen to hydrogen peroxide.

As used herein, the term “choline oxidase” (“COx”) refers to an oxidaseenzyme (EC 1.1.3.17) that catalyzes the four-electron oxidation ofcholine to glycine betaine, with betaine aldehyde as an intermediatewith concomitant reduction of two molecules of molecular oxygen to twomolecules of hydrogen peroxide.

As used herein, the term “hexose oxidase” (“HOx”) refers to an oxidaseenzyme (EC 1.1.3.5) that is capable of the oxidation of mono- anddisaccharides to their corresponding lactones, with concomitantreduction of molecular oxygen to hydrogen peroxide. Hexose oxidase isable to oxidize a variety of substrates including D-glucose,D-galactose, maltose, cellobiose, and lactose, etc. It is not intendedthat the present invention be limited to any particular hexose.

As used herein, “glycerol oxidase” refers to an oxidase enzyme (EC1.1.3.) that catalyzes the oxidation of glycerol to glyceraldehyde, withconcomitant reduction of molecular oxygen to hydrogen peroxide.

As used herein, “sorbitol oxidase” or “SOx” refers to a polyol oxidaseenzyme (EC 1.1.3.) that catalyzes the oxidation of a substrate (e.g.,D-sorbitol) to D-glucose, with concomitant reduction of molecular oxygento hydrogen peroxide. The definition further includes oxidase enzymeswith a Vmax/Km value which is highest for sorbitol than for xylitol,galactitol, D-mannitol, glycerol and D-arabitol, and no significantactivity towards xylose, D-glucose, galactose, mannose and arabinose.

As used herein, the term “xylitol oxidase” (“XOx”) refers to an oxidaseenzyme that catalyzes the oxidation of xylitol to xylose withconcomitant reduction of molecular oxygen to hydrogen peroxide. Thedefinition further includes oxidase enzymes with a Vmax/Km value whichis highest for xylitol than for sorbitol, galactitol, D-mannitol, andD-arabitol, and no activity towards xylose, D-glucose, galactose,mannose and arabinose.

In some particularly preferred embodiments, the sorbitol oxidase of thepresent invention has a higher specific activity, (or V_(max)/K_(km),ratio) on sorbitol substrate, as compared to an alternative substrateunder standard assay conditions (e.g, in the in vitro assay providedbelow) and/or using an in situ in an application media, conducted asknown in the art. In some preferred alternative embodiments, thealternative substrate is xylitol.

In some preferred embodiments, the following in vitro assay finds use.An assay mixture containing 266 ul substrate (e.g., sorbitol—SigmaP-5504 or xylitol) (0.055 M, in 0.1 M sodium phosphate buffer, pH 6.3),12 ul 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS)(Sigma A-9941, 5 mg/ml aqueous solution), 12 ul peroxidase (POD) (SigmaP-6782, 0.1 mg/ml in 0.1 M sodium phosphate buffer, pH 6.3) and 10 ulenzyme (SOx or HOx) aqueous solution was prepared. The assay wasperformed at 25° C. The incubation was started by the addition ofglucose to the assay mixture. The absorbance was monitored at 405 nm inan ELISA reader. A standard curve, based on varying concentrations ofH₂O₂, was used for calculation of enzyme activity. In this assay, 1polyol unit (POx), such as SOx or XOX units, corresponds to the amountof enzyme, which under the specified conditions results in theconversion of 1 umole of the specified polyol per minute, with resultantgeneration of 1 umole of hydrogen peroxide (H₂O₂).

In some particularly preferred embodiments, the sorbitol oxidase of thepresent invention has no significant activity on the corresponding sugarproduct, such as glucose, xylose, galactose, mannose or arabinose,preferably glucose as determined using the assay provided in theExamples.

In some alternative preferred embodiments, the xylitol oxidase of thepresent invention has a higher specific activity, (or V_(max)/K_(km),ratio) on xylitol substrate, as compared to sorbitol under standardassay conditions (e.g., in the in vitro assay provided herein) and/or inan in situ in an application media, conducted as known in the art.

As used herein, “inhibitors” refers to chemical compounds that canreduce or stop the catalytic activity of an enzyme. In particularlypreferred embodiments, the inhibitors reduce or stop the catalyticactivity of at least one oxidase. Examples of oxidase inhibitors includeacetate, silver salts, halide ions, sec- and tert-alcohols, isocyanate,isothiocyante, glucose analogs, bisulfite, sulfite, thiosulfate,metabisulfite, zinc salts, diethyl dicarbamate, methyl methanesulfonate, acrylonitrile, 2-amino, 2-methyl 1-propanol.

As used herein, “reversible enzyme inhibitor” refers to molecules thatbind to an enzyme and decrease its rate of reaction. In someembodiments, reversible enzyme inhibitors are affected by varying theconcentration of the enzyme's substrate in relation to the inhibitor. Insome embodiments, reversible enzyme inhibitors bind to the enzyme usingweak bonds that are similar to those used to bind to substrate. Thus,the reversible inhibitor does not permanently disable the enzyme, asremoval of the inhibitor allows the enzyme to bind to and turnover itssubstrate. In some embodiments, reversible enzyme inhibitors arecompetitive inhibitors that interact non-covalently with the enzyme,and/or compete with the substrate for the enzyme's active site, and/orhave structures that are similar to the substrate, products and/ortransition state. In additional embodiments, the reversible inhibitor isa non-competitive enzyme inhibitor that binds at a site present on theenzyme other than the active site, and/or causes conformational changesin the enzyme that decrease, and/or stop catalytic activity. It is notintended that the term be limited to any particular mechanism or type ofreversible enzyme inhibitor. It is only necessary that the effects ofthe enzyme inhibitor be reversible, such that the enzyme will functionin the absence of the inhibitor and/or dilutions of the enzyme inhibitormixture.

As used herein, the term “compatible,” means that other compositionmaterials (i.e., other materials in a given composition, in addition toan oxidase of the present invention) do not reduce the enzymaticactivity of the oxidase enzyme(s) provided herein to such an extent thatthe oxidases(s) is/are not effective as desired during normal usesituations. Specific composition materials are exemplified in detailhereinafter.

As used herein, “effective amount of enzyme” refers to the quantity ofenzyme necessary to achieve the enzymatic activity required in thespecific application. Such effective amounts are readily ascertained byone of ordinary skill in the art and are based on many factors, such asthe particular enzyme variant used, the application, the specificcontents of the composition, and whether a liquid or dry (e.g.,granular) composition is required, and the like.

As used herein, “having improved properties” used in connection with“mutant oxidative enzymes,” refers to oxidative enzymes with improvedperformance and/or improved stability with retained performance,relative to the corresponding wild-type oxidase. In some particularlypreferred embodiments, the improved properties are selected from thegroup consisting of improved dishwash performance and improvedstability, as well as the combination of improved dishwash performanceand improved stability.

As used herein, the phrase “detergent stability” refers to the stabilityof a detergent composition. In some embodiments, the stability isassessed during the use of the detergent, while in other embodiments,the term refers to the stability of a detergent composition duringstorage.

The term “improved stability” is used to indicate better stability ofoxidases combined with mutant protease(s) in compositions during storageand/or better stability during use (e.g., in the sud). In preferredembodiments, the mutant oxidases combined with mutant protease(s)exhibit improved stability in formulations during storage and/orimproved stability during use, which includes stability againstoxidizing agents, sequestering agents, autolysis, surfactants and highalkalinity, relative to the corresponding wild-type enzyme.

As used herein, “oxidative stability” refers to the ability of a proteinto function under oxidative conditions. In particular, the term refersto the ability of a protein to function in the presence of variousconcentrations of H₂O₂, peracids and other oxidants. Stability undervarious oxidative conditions can be measured either by standardprocedures known to those in the art and/or by the methods describedherein. A substantial change in oxidative stability is evidenced by atleast about a 5% or greater increase or decrease (in most embodiments,it is preferably an increase) in the half-life of the enzymaticactivity, as compared to the enzymatic activity present in the absenceof oxidative compounds.

As used herein, “pH stability” refers to the ability of a protein tofunction at a particular pH. In general, most enzymes have a finite pHrange at which they will function. In addition to enzymes that functionin mid-range pHs (i.e., around pH 7), there are enzymes that are capableof working under conditions with very high or very low pHs. Stability atvarious pHs can be measured either by standard procedures known to thosein the art and/or by the methods described herein. A substantial changein pH stability is evidenced by at least about 5% or greater increase ordecrease (in most embodiments, it is preferably an increase) in thehalf-life of the enzymatic activity, as compared to the enzymaticactivity at the enzyme's optimum pH. However, it is not intended thatthe present invention be limited to any pH stability level nor pH range.

As used herein, “thermal stability” refers to the ability of a proteinto function at a particular temperature. In general, most enzymes have afinite range of temperatures at which they will function. In addition toenzymes that work in mid-range temperatures (e.g., room temperature),there are enzymes that are capable of working in very high or very lowtemperatures. Thermal stability can be measured either by knownprocedures or by the methods described herein. A substantial change inthermal stability is evidenced by at least about 5% or greater increaseor decrease (in most embodiments, it is preferably an increase) in thehalf-life of the catalytic activity of a mutant when exposed to giventemperature However, it is not intended that the present invention belimited to any temperature stability level nor temperature range.

As used herein, the term “chemical stability” refers to the stability ofa protein (e.g., an enzyme) towards chemicals that may adversely affectits activity. In some embodiments, such chemicals include, but are notlimited to hydrogen peroxide, peracids, anionic detergents, cationicdetergents, non-ionic detergents, chelants, etc. However, it is notintended that the present invention be limited to any particularchemical stability level nor range of chemical stability.

As used herein, the terms “purified” and “isolated” refer to the removalof contaminants from a sample. For example, polyol oxidases are purifiedby removal of contaminating proteins and other compounds within asolution or preparation that are not polyol oxidases. In someembodiments, recombinant polyol oxidases are expressed in bacterial orfungal host cells and these recombinant polyol oxidases are purified bythe removal of other host cell constituents; the percent of recombinantpolyol oxidase polypeptides is thereby increased in the sample. Inparticularly preferred embodiments, the polyol oxidases of the presentinvention are substantially purified to a level of at least about 99% ofthe protein component, as determined by SDS-PAGE or other standardmethods known in the art.

As used herein, “protein of interest” and “polypeptide of interest”refer to a protein (e.g., an enzyme or “enzyme of interest”) which isbeing analyzed, identified and/or modified. Naturally-occurring, as wellas recombinant proteins find use in the present invention.

As used herein, “protein” refers to any composition comprised of aminoacids and recognized as a protein by those of skill in the art. Theterms “protein,” “peptide” and polypeptide are used interchangeablyherein. Wherein a peptide is a portion of a protein, those skilled inthe art understand the use of the term in context.

The terms “mature form” and “mature region” refer to the finalfunctional portion of the protein. To exemplify, a mature form of thePOx of the present invention at least includes the amino acid sequenceidentical to SEQ ID NO:2, SEQ ID NO:4 and/or SEQ ID NO:6. In thiscontext, the “mature form” is “processed from” a full-length POx,wherein the processing of the full-length carrageenase encompasses theremoval of the signal peptide. Thus, for example, the mature form of thePOx of SEQ ID NO:2 corresponds to amino acid residues 48 to 467 of SEQID NO:16; and the mature form of the POx of SEQ ID NO:6 corresponds toamino acid residues 28 to 480 of SEQ ID NO:37.

A “full-length” protein herein refers to a POx polypeptide thatcomprises a secretory signal peptide and a mature portion. The fusionPOx enzymes of the invention are encoded by chimeric polynucleotides.

The term “chimeric polynucleotide” or “fusion polynucleotide” hereinrefer to a polynucleotide that comprises at least two separate anddistinct regions that may or may not originate from the same gene. Forexample, a polynucleotide sequence encoding a signal peptide linked tothe polynucleotide that encodes the mature form of the polypeptide wouldbe termed a chimeric polynucleotide. In some embodiments, a chimeric POxpolynucleotide encodes a POx fusion polypeptide.

As used herein, the terms “chimeric polypeptide” and “fusionpolypeptide” are used interchangeably to refer to a protein thatcomprises at least two separate and distinct regions that may or may notoriginate from the same protein. For example, a signal peptide linked tothe protein of interest wherein the signal peptide is not normallyassociated with the protein of interest would be termed a chimericpolypeptide or chimeric protein. A “POx fusion polypeptide” or “POxchimeric polypeptide” herein refers to a polypeptide comprising thesequence of the mature form of the POx enzyme operably linked to a POxsignal peptide.

The terms “signal sequence,” “signal peptide” and “secretory signalpeptide” refer to any sequence of nucleotides and/or amino acids whichmay participate in the production of the mature or fusion forms of theprotein. This definition of signal sequence is a functional one, meantto include all those amino acid sequences encoded by the N-terminalportion of the protein gene, which participate in the effectuation ofthe secretion of the protein. A “POx signal peptide” herein refers to asignal peptide that is linked to the N-terminus of the mature form ofthe POx enzyme.

“Naturally-occurring” or “wild-type” refers to a POx protein or apolynucleotide encoding a POx protein having the unmodified amino acidsequence identical to that found in nature. Naturally occurring enzymesinclude native enzymes, such as those enzymes naturally expressed orfound in the particular microorganism. A sequence that is wild-type ornaturally-occurring refers to a sequence from which a variant, or asynthetic sequence is derived. The wild-type sequence may encode eithera homologous or heterologous protein.

As used herein the term “expression” refers to a process by which apolynucleotide is transcribed and the resulting transcript is translatedto yield a polypeptide.

As used herein, functionally and/or structurally similar proteins areconsidered to be “related proteins.” In some embodiments, these proteinsare enzymatically active on polyols. In some embodiments, these proteinsare derived from a different genus and/or species, including differencesbetween classes of organisms (e.g., a bacterial protein and a fungalprotein). In some embodiments, these proteins are derived from adifferent genus and/or species, including differences between classes oforganisms (e.g., a bacterial enzyme and a fungal enzyme). In additionalembodiments, related proteins are provided from the same species.Indeed, it is not intended that the present invention be limited torelated proteins from any particular source(s). In addition, the term“related proteins” encompasses tertiary structural homologs and primarysequence homologs (e.g., the polyol oxidases of the present invention).In further embodiments, the term encompasses proteins that areimmunologically cross-reactive.

As used herein, the term “derivative” refers to a protein which isderived from a protein by addition of one or more amino acids to eitheror both the C- and N-terminal end(s), substitution of one or more aminoacids at one or a number of different sites in the amino acid sequence,and/or deletion of one or more amino acids at either or both ends of theprotein or at one or more sites in the amino acid sequence, and/orinsertion of one or more amino acids at one or more sites in the aminoacid sequence. The preparation of a protein derivative is preferablyachieved by modifying a DNA sequence which encodes for the nativeprotein, transformation of that DNA sequence into a suitable host, andexpression of the modified DNA sequence to form the derivative protein.

Related (and derivative) proteins comprise “variant proteins.” In somepreferred embodiments, variant proteins differ from a parent protein andone another by a small number of amino acid residues. In someparticularly preferred embodiments, related proteins and particularlyvariant proteins comprise at least about 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or 99% amino acid sequence identity. Additionally, arelated protein or a variant protein as used herein, refers to a proteinthat differs from another related protein or a parent protein in thenumber of prominent regions. For example, in some embodiments, variantproteins have 1, 2, 3, 4, 5, or 10 corresponding prominent regions thatdiffer from the parent protein.

In some embodiments, amino acid and polynucleotide homology isdetermined using methods known in the art (e.g., the ClustalW algorithm,using standard settings, with EMBOSS::water (local): Gap Open=10.0, Gapextend=0.5, using Blosum 62 (protein), or DNA full for nucleotidesequences.)

As used herein, the term “variant(s)” as used in context of apolypeptide sequence (e.g., SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 16, 28, 33, 35, 37, 38, and/or 40 refers to a polypeptideprepared from the original (i.e., parent) polypeptide, or by using thesequence information from the original (i.e., parent) polypeptide, byinsertion, deletion and/or substitution of one or more amino acids inthe original (i.e., parent) sequence. In some embodiments, at least oneinsertion, deletion, and/or substitution is made in the original (i.e.,parent) sequence, while in other embodiments, preferably less than about50 amino acids, less than about 40, less than about 30, less than about20, or less than about 10 amino acids are modified by insertion,deletion and/or substitution of the original (i.e., parent) sequence inorder to produce variant polypeptides. In some preferred embodiments,only one amino acid modification (i.e., insertion, deletion orsubstitution) is made, while in other preferred embodiments two aminoacids are modified, and in still further embodiments, three amino acidsare modified, and in yet additional embodiments, four amino acids aremodified, and in still further embodiments, five amino acids aremodified. It is not intended that the variants of the present inventionbe limited to any specific number nor type of amino acid modifications.

The terms “precursor” or “parent” polypeptide herein refer to thepolypeptide that was modified to provide a variant polypeptide.

As used herein, the term “homologue(s),” when used herein in the contextof a polypeptide sequence (e.g., SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 16, 28, 33, 35, 37, 38, and/or 40) refers to a polypeptidewhich is at least about 70% homologous, more preferably at least about80% homologous, still more preferably at least about 85% homologous,further more preferably at least about 90% homologous, even morepreferably at least about 95%, more preferably about 96%, still morepreferably about 97%, even more preferably about 98%, or most preferablyabout 99% homologous to the polypeptide sequence of interest (e.g., apolyol oxidase provided by the present invention). In some embodiments,homology between two polypeptide sequences is determined using ClustalWalignment algorithm using standard settings, as referred to herein.However, it is not intended that the present invention be limited to anyparticular method for determining homology.

The term “heterologous” used in this context refers to a sequence whichoriginates from a species or strain other than the species or strainfrom which the polypeptide, or the parent polypeptide from which thepolypeptide is derived (i.e., a variant, homologue or fragment) isnaturally found, for example a heterologous signal peptide encodingpolynucleotide.

As used herein, the term “fragment(s).” as used herein in the context ofa polypeptide sequence (e.g., SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 16, 28, 33, 35, 37, 38, and/or 40) refers to a polypeptidewhich consists of only a part of the original (i.e., parent) polypeptidesequence. In some embodiments, fragments comprise at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95% of the original (i.e., parent) polypeptidesequence.

The variants, homologues and fragments provided by the present inventionall retain at least some of the desired enzymatic activity of the parentenzyme, such as at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80% at least about 90%, or all theenzyme activity of the parent enzyme. In some alternative embodiments,the variants and/or homologues have greater enzymatic activity than theoriginal (i.e., parent) enzyme.

It is intended that although preferred enzymes for use in the vectorsand methods of the invention are referred to herein by their specificSEQ ID NOS, the present invention encompasses enzymes which are derivedfrom the nucleic acids which encode the corresponding amino acid SEQ IDNOS, when expressed, either in their native host species or aheterologous host species. Thus, the present invention encompassesembodiments in which the enzymes are co- or post-translationallyprocessed.

Several methods are known in the art that are suitable for generatingvariants of the enzymes of the present invention, including but notlimited to site-saturation mutagenesis, scanning mutagenesis,insertional mutagenesis, random mutagenesis, site-directed mutagenesis,and directed-evolution, as well as various other recombinatorialapproaches.

Characterization of wild-type and mutant proteins is accomplished viaany means suitable and is preferably based on the assessment ofproperties of interest. For example, pH and/or temperature, as well asdetergent and/or oxidative stability is/are determined in someembodiments of the present invention. Indeed, it is contemplated thatenzymes having various degrees of stability in one or more of thesecharacteristics (pH, temperature, proteolytic stability, detergentstability, and/or oxidative stability) will find use.

As used herein, “expression vector” refers to a DNA construct containinga DNA sequence that is operably linked to a suitable control sequencecapable of effecting the expression of the DNA in a suitable host. Suchcontrol sequences include a promoter to effect transcription, anoptional operator sequence to control such transcription, a sequenceencoding suitable mRNA ribosome binding sites and sequences whichcontrol termination of transcription and translation. The vector may bea plasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, “plasmid,”“expression plasmid,” and “vector” are often used interchangeably as theplasmid is the most commonly used form of vector at present. However,the invention is intended to include such other forms of expressionvectors that serve equivalent functions and which are, or become, knownin the art.

In some preferred embodiments, the POx gene is ligated into anappropriate expression plasmid. The cloned POx gene is then used totransform or transfect a host cell in order to express the sorbitoloxidase gene. This plasmid may replicate in hosts in the sense that itcontains the well-known elements necessary for plasmid replication orthe plasmid may be designed to integrate into the host chromosome. Thenecessary elements are provided for efficient gene expression (e.g., apromoter operably linked to the gene of interest). In some embodiments,these necessary elements are supplied as the gene's own homologouspromoter if it is recognized, (i.e., transcribed, by the host), atranscription terminator (a polyadenylation region for eukaryotic hostcells) which is exogenous or is supplied by the endogenous terminatorregion of the sorbitol oxidase gene. In some embodiments, a selectiongene such as an antibiotic resistance gene that enables continuouscultural maintenance of plasmid-infected host cells by growth inantimicrobial-containing media is also included.

As used herein, “corresponding to,” refers to a residue at theenumerated position in a protein or peptide, or a residue that isanalogous, homologous, or equivalent to an enumerated residue in aprotein or peptide.

As used herein, “corresponding region,” generally refers to an analogousposition along related proteins or a parent protein.

The terms “nucleic acid molecule encoding,” “nucleic acid sequenceencoding,” “DNA sequence encoding,” and “DNA encoding” refer to theorder or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (protein)chain. The DNA sequence thus codes for the amino acid sequence.

The term “POx polynucleotide” herein refers to the DNA sequence encodinga POx polypeptide.

As used herein, the term “analogous sequence” refers to a sequencewithin a protein that provides similar function, tertiary structure,and/or conserved residues as the protein of interest (i.e., typicallythe original protein of interest). For example, in epitope regions thatcontain an alpha helix or a beta sheet structure, the replacement aminoacids in the analogous sequence preferably maintain the same specificstructure. The term also refers to nucleotide sequences, as well asamino acid sequences. In some embodiments, analogous sequences aredeveloped such that the replacement amino acids result in a variantenzyme showing a similar or improved function. In some preferredembodiments, the tertiary structure and/or conserved residues of theamino acids in the protein of interest are located at or near thesegment or fragment of interest. Thus, where the segment or fragment ofinterest contains, for example, an alpha-helix or a beta-sheetstructure, the replacement amino acids preferably maintain that specificstructure.

As used herein, “homologous protein” refers to a protein (e.g., polyoloxidase) that has similar action and/or structure, as a protein ofinterest (e.g., a polyol oxidase from another source). It is notintended that homologs be necessarily related evolutionarily. Thus, itis intended that the term encompass the same or similar enzyme(s) (i.e.,in terms of structure and function) obtained from different species. Insome preferred embodiments, it is desirable to identify a homolog thathas a quaternary, tertiary and/or primary structure similar to theprotein of interest, as replacement for the segment or fragment in theprotein of interest with an analogous segment from the homolog willreduce the disruptiveness of the change. In some preferred embodiments,the homologs are selected from SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11,12 and 13, while in other embodiments, the homolog is SEQ ID NO:14.

As used herein, “homologous genes” refers to at least a pair of genesfrom different species, which genes correspond to each other and whichare identical or very similar to each other. The term encompasses genesthat are separated by speciation (i.e., the development of new species)(e.g., orthologous genes), as well as genes that have been separated bygenetic duplication (e.g., paralogous genes). These genes encode“homologous proteins.”

As used herein, “ortholog” and “orthologous genes” refer to genes indifferent species that have evolved from a common ancestral gene (i.e.,a homologous gene) by speciation. Typically, orthologs retain the samefunction during the course of evolution. Identification of orthologsfinds use in the reliable prediction of gene function in newly sequencedgenomes.

As used herein, “paralog” and “paralogous genes” refer to genes that arerelated by duplication within a genome. While orthologs retain the samefunction through the course of evolution, paralogs evolve new functions,even though some functions are often related to the original one.

As used herein, “wild-type” and “native” proteins are those found innature. The terms “wild-type sequence,” and “wild-type gene” are usedinterchangeably herein, to refer to a sequence that is native ornaturally occurring in a host cell. In some embodiments, the wild-typesequence refers to a sequence of interest (i.e., a sequence that isbeing analyzed, assessed, modified, etc.) that is the starting point ofa protein engineering project. The genes encoding thenaturally-occurring protein may be obtained in accord with the generalmethods known to those skilled in the art. The methods generallycomprise synthesizing labeled probes having putative sequences encodingregions of the protein of interest, preparing genomic libraries fromorganisms expressing the protein, and screening the libraries for thegene of interest by hybridization to the probes. Positively hybridizingclones are then mapped and sequenced.

The term “recombinant DNA molecule” as used herein refers to a DNAmolecule that is comprised of segments of DNA joined together by meansof molecular biological techniques.

The term “recombinant oligonucleotide” refers to an oligonucleotidecreated using molecular biological manipulations, including but notlimited to, the ligation of two or more oligonucleotide sequencesgenerated by restriction enzyme digestion of a polynucleotide sequence,the synthesis of oligonucleotides (e.g., the synthesis of primers oroligonucleotides) and the like.

The degree of homology between sequences may be determined using anysuitable method known in the art (See e.g., Smith and Waterman, Adv.Appl. Math., 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol., 48:443[1970]; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988];programs such as GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package (Genetics Computer Group, Madison, Wis.); andDevereux et al., Nucl. Acid Res., 12:387-395 [1984]).

For example, PILEUP is a useful program to determine sequence homologylevels. PILEUP creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments. It can alsoplot a tree showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng and Doolittle, (Feng and Doolittle, J. Mol. Evol.,35:351-360 [1987]). The method is similar to that described by Higginsand Sharp (Higgins and Sharp, CABIOS 5:151-153 [1989]). Useful PILEUPparameters including a default gap weight of 3.00, a default gap lengthweight of 0.10, and weighted end gaps. Another example of a usefulalgorithm is the BLAST algorithm, described by Altschul et al.,(Altschul et al., J. Mol. Biol., 215:403-410, [1990]; and Karlin et al.,Proc. Natl. Acad. Sci. USA 90:5873-5787 [1993]). One particularly usefulBLAST program is the WU-BLAST-2 program (See, Altschul et al., Meth.Enzymol., 266:460-480 [1996]). parameters “W,” “T,” and “X” determinethe sensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 [1989])alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

As used herein, “percent (%) nucleic acid sequence identity” is definedas the percentage of nucleotide residues in a candidate sequence that isidentical with the nucleotide residues of the sequence.

As used herein, the term “hybridization” refers to the process by whicha strand of nucleic acid joins with a complementary strand through basepairing, as known in the art.

As used herein, the phrase “hybridization conditions” refers to theconditions under which hybridization reactions are conducted. Theseconditions are typically classified by degree of “stringency” of theconditions under which hybridization is measured. The degree ofstringency can be based, for example, on the melting temperature (Tm) ofthe nucleic acid binding complex or probe. For example, “maximumstringency” typically occurs at about Tm-5° C. (5° below the Tm of theprobe); “high stringency” at about 5-10° below the Tm; “intermediatestringency” at about 10-20° below the Tm of the probe; and “lowstringency” at about 20-25° below the Tm. Alternatively, or in addition,hybridization conditions can be based upon the salt or ionic strengthconditions of hybridization and/or one or more stringency washes. Forexample, 6×SSC=very low stringency; 3×SSC=low to medium stringency;1×SSC=medium stringency; and 0.5×SSC=high stringency. Functionally,maximum stringency conditions may be used to identify nucleic acidsequences having strict identity or near-strict identity with thehybridization probe; while high stringency conditions are used toidentify nucleic acid sequences having about 80% or more sequenceidentity with the probe. For applications requiring high selectivity, itis typically desirable to use relatively stringent conditions to formthe hybrids (e.g., relatively low salt and/or high temperatureconditions are used).

The phrases “substantially similar and “substantially identical” in thecontext of at least two nucleic acids or polypeptides typically meansthat a polynucleotide or polypeptide comprises a sequence that has atleast about 40% identity, more preferable at least about 50% identity,yet more preferably at least about 60% identity, preferably at leastabout 75% identity, more preferably at least about 80% identity, yetmore preferably at least about 90%, still more preferably about 95%,most preferably about 97% identity, sometimes as much as about 98% andabout 99% sequence identity, compared to the reference (i.e., wild-type)sequence. Sequence identity may be determined using known programs suchas BLAST, ALIGN, and CLUSTAL using standard parameters. (See e.g.,Altschul, et al., J. Mol. Biol. 215:403-410 [1990]; Henikoff et al.,Proc. Natl. Acad. Sci. USA 89:10915 [1989]; Karin et al., Proc. Natl.Acad. Sci. USA 90:5873 [1993]; and Higgins et al., Gene 73:237-244[1988]). Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information. Also,databases may be searched using FASTA (Pearson et al., Proc. Natl. Acad.Sci. USA 85:2444-2448 [1988]). One indication that two polypeptides aresubstantially identical is that the first polypeptide is immunologicallycross-reactive with the second polypeptide. Typically, polypeptides thatdiffer by conservative amino acid substitutions are immunologicallycross-reactive. Thus, a polypeptide is substantially identical to asecond polypeptide, for example, where the two peptides differ only by aconservative substitution. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions (e.g., within a rangeof medium to high stringency).

As used herein, “equivalent residues” refers to proteins that shareparticular amino acid residues. For example, equivalent resides may beidentified by determining homology at the level of tertiary structurefor a protein (e.g., polyol oxidase) whose tertiary structure has beendetermined by x-ray crystallography. Equivalent residues are defined asthose for which the atomic coordinates of two or more of the main chainatoms of a particular amino acid residue of the protein having putativeequivalent residues and the protein of interest (N on N, CA on CA, C onC and O on O) are within 0.13 nm and preferably 0.1 nm after alignment.Alignment is achieved after the best model has been oriented andpositioned to give the maximum overlap of atomic coordinates ofnon-hydrogen protein atoms of the proteins analyzed. The preferred modelis the crystallographic model giving the lowest R factor forexperimental diffraction data at the highest resolution available,determined using methods known to those skilled in the art ofcrystallography and protein characterization/analysis.

As used herein, the terms “hybrid polyol oxidases” and “fusion polyoloxidases” refer to proteins that are engineered from at least twodifferent or “parental” proteins. In preferred embodiments, theseparental proteins are homologs of one another. For example, in someembodiments, a preferred hybrid sequence or fusion protein contains theN-terminus of a protein and the C-terminus of a homolog of the protein.In some preferred embodiment, the two terminal ends are combined tocorrespond to the full-length active protein.

The term “regulatory element” as used herein refers to a genetic elementthat controls some aspect of the expression of nucleic acid sequences.For example, a promoter is a regulatory element which facilitates theinitiation of transcription of an operably linked coding region.Additional regulatory elements include splicing signals, polyadenylationsignals and termination signals.

As used herein, “host cells” are generally prokaryotic or eukaryotichosts which are transformed or transfected with vectors constructedusing recombinant DNA techniques known in the art. Transformed hostcells are capable of either replicating vectors encoding the proteinvariants or expressing the desired protein variant. In the case ofvectors which encode the pre- or prepro-form of the protein variant,such variants, when expressed, are typically secreted from the host cellinto the host cell medium.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means transformation, transduction ortransfection. Means of transformation include protoplast transformation,calcium chloride precipitation, electroporation, naked DNA and the likeas known in the art. (See, Chang and Cohen, Mol. Gen. Genet.,168:111-115 [1979]; Smith et al., Appl. Env. Microbiol., 51:634 [1986];and the review article by Ferrari et al., in Harwood, Bacillus, PlenumPublishing Corporation, pp. 57-72 [1989]).

The term “promoter/enhancer” denotes a segment of DNA which containssequences capable of providing both promoter and enhancer functions (forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions). The enhancer/promoter may be “endogenous” or“exogenous” or “heterologous.” An endogenous enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An exogenous(heterologous) enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques).

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichhas stably integrated foreign or exogenous DNA into the genomic DNA ofthe transfected cell.

The terms “selectable marker” or “selectable gene product” as usedherein refer to the use of a gene which encodes an enzymatic activitythat confers resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed.

As used herein, the terms “amplification” and “gene amplification” referto a process by which specific DNA sequences are disproportionatelyreplicated such that the amplified gene becomes present in a higher copynumber than was initially present in the genome. In some embodiments,selection of cells by growth in the presence of a drug (e.g., aninhibitor of an inhibitable enzyme) results in the amplification ofeither the endogenous gene encoding the gene product required for growthin the presence of the drug or by amplification of exogenous (i.e.,input) sequences encoding this gene product, or both. Selection of cellsby growth in the presence of a drug (e.g., an inhibitor of aninhibitable enzyme) may result in the amplification of either theendogenous gene encoding the gene product required for growth in thepresence of the drug or by amplification of exogenous (i.e., input)sequences encoding this gene product, or both.

“Amplification” is a special case of nucleic acid replication involvingtemplate specificity. It is to be contrasted with non-specific templatereplication (i.e., replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

As used herein, the term “co-amplification” refers to the introductioninto a single cell of an amplifiable marker in conjunction with othergene sequences (i.e., comprising one or more non-selectable genes suchas those contained within an expression vector) and the application ofappropriate selective pressure such that the cell amplifies both theamplifiable marker and the other, non-selectable gene sequences. Theamplifiable marker may be physically linked to the other gene sequencesor alternatively two separate pieces of DNA, one containing theamplifiable marker and the other containing the non-selectable marker,may be introduced into the same cell.

As used herein, the terms “amplifiable marker,” “amplifiable gene,” and“amplification vector” refer to a marker, gene or a vector encoding agene which permits the amplification of that gene under appropriategrowth conditions.

As used herein, the term “amplifiable nucleic acid” refers to nucleicacids which may be amplified by any amplification method. It iscontemplated that “amplifiable nucleic acid” will usually comprise“sample template.”

As used herein, the term “sample template” refers to nucleic acidoriginating from a sample which is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

“Template specificity” is achieved in most amplification techniques bythe choice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase (See e.g., Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038[1972]). Other nucleic acids are not replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters (See,Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase,the enzyme will not ligate the two oligonucleotides or polynucleotides,where there is a mismatch between the oligonucleotide or polynucleotidesubstrate and the template at the ligation junction (See, Wu andWallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, byvirtue of their ability to function at high temperature, are found todisplay high specificity for the sequences bounded and thus defined bythe primers; the high temperature results in thermodynamic conditionsthat favor primer hybridization with the target sequences and nothybridization with non-target sequences.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. It is not intended that the present invention be limited to anyparticular detection system or label.

As used herein, the term “target,” when used in reference toamplification methods (e.g., the polymerase chain reaction), refers tothe region of nucleic acid bounded by the primers used for polymerasechain reaction. Thus, the “target” is sought to be sorted out from othernucleic acid sequences. A “segment” is defined as a region of nucleicacid within the target sequence.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188,hereby incorporated by reference, which include methods for increasingthe concentration of a segment of a target sequence in a mixture ofgenomic DNA without cloning or purification. This process for amplifyingthe target sequence consists of introducing a large excess of twooligonucleotide primers to the DNA mixture containing the desired targetsequence, followed by a precise sequence of thermal cycling in thepresence of a DNA polymerase. The two primers are complementary to theirrespective strands of the double stranded target sequence. To effectamplification, the mixture is denatured and the primers then annealed totheir complementary sequences within the target molecule. Followingannealing, the primers are extended with a polymerase so as to form anew pair of complementary strands. The steps of denaturation, primerannealing and polymerase extension can be repeated many times (i.e.,denaturation, annealing and extension constitute one “cycle”; there canbe numerous “cycles”) to obtain a high concentration of an amplifiedsegment of the desired target sequence. The length of the amplifiedsegment of the desired target sequence is determined by the relativepositions of the primers with respect to each other, and therefore, thislength is a controllable parameter. By virtue of the repeating aspect ofthe process, the method is referred to as the “polymerase chainreaction” (hereinafter “PCR”). Because the desired amplified segments ofthe target sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

As used herein, the term “amplification reagents” refers to thosereagents (deoxyribonucleotide triphosphates, buffer, etc.), needed foramplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTPor dATP, into the amplified segment). In addition to genomic DNA, anyoligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process itself are, themselves, efficienttemplates for subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

“Recovering” a polypeptide from a culture medium refers to collectingthe polypeptide in the culture medium into which it was secreted by thehost cell. The polypeptide can also be recovered from a lysate preparedfrom the host cells and further purified. A secreted polypeptide may berecovered from the cell wall fraction prepared according to methodsknown in the art. One skilled in the art can readily follow knownmethods for isolating polypeptides and proteins in order to obtain oneof the isolated polypeptides or proteins of the present invention. Theseinclude, but are not limited to, immunochromatography, HPLC,size-exclusion chromatography, ion-exchange chromatography, andimmuno-affinity chromatography. See, e.g., Scopes, Protein Purification:Principles and Practice, Springer-Verlag (1994); Sambrook, et al., inMolecular Cloning: A Laboratory Manual; Ausubel et al., CurrentProtocols in Molecular Biology. The protein produced by a recombinanthost cell comprising a secretion factor of the present invention will besecreted into the culture media.

As used herein, “compositions”, “cleaning compositions” and “cleaningformulations” refer to compositions that find use in the removal ofundesired compounds from items to be cleaned, such as fabric, dishes,contact lenses, other solid substrates, hair (shampoos), skin (soaps andcreams), teeth (mouthwashes, toothpastes) etc. The term encompasses anymaterials/compounds selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, gel,granule, or spray composition), as long as the composition is compatiblewith the oxidase and other enzyme(s) used in the composition, and anyreversible enzyme inhibitors in the composition. The specific selectionof cleaning composition materials are readily made by considering thesurface, item or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use.

The terms further refer to any composition that is suited for cleaning,bleaching, disinfecting, and/or sterilizing any object and/or surface.It is intended that the terms include, but are not limited to detergentcompositions (e.g., liquid and/or solid laundry detergents and finefabric detergents; hard surface cleaning formulations, such as forglass, wood, ceramic and metal counter tops and windows; carpetcleaners; oven cleaners; fabric fresheners; fabric softeners; andtextile and laundry pre-spotters, as well as dish detergents).

Indeed, the term “cleaning composition” as used herein, includes unlessotherwise indicated, granular or powder-form all-purpose or heavy-dutywashing agents, especially cleaning detergents; liquid, gel orpaste-form all-purpose washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, mouthwashes, denturecleaners, car or carpet shampoos, bathroom cleaners; hair shampoos andhair-rinses; shower gels and foam baths and metal cleaners; as well ascleaning auxiliaries such as bleach additives and “stain-stick” orpre-treat types.

As used herein, the terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In somepreferred embodiments, the term is used in reference to launderingfabrics and/or garments (e.g., “laundry detergents”). In alternativeembodiments, the term refers to other detergents, such as those used toclean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is notintended that the present invention be limited to any particulardetergent formulation or composition. Indeed, it is intended that inaddition to oxidase, the term encompasses detergents that containsurfactants, transferase(s), hydrolytic enzymes, oxido reductases,perhydrolases builders, bleaching agents, bleach activators, bluingagents and fluorescent dyes, caking inhibitors, masking agents, enzymeactivators, enzyme inhibitors, antioxidants, and solubilizers. In somepreferred embodiments, the detergent formulations include, but are notlimited to those set forth in U.S. patent application Ser. Nos.10/576,331 and 10/581,014, as well as WO 05/52161 and WO 05/056782 finduse in the present invention. However, it is not intended that thepresent invention be limited to any particular detergent formulation(s),as any suitable detergent formulation finds use in the presentinvention.

As used herein, “textile materials” is a general term for fibers, yarnintermediates, yarn, fabrics, and products made from fabrics (e.g.,garments and other articles).

As used herein, “fabric” encompasses any textile material. Thus, it isintended that the term encompass garments, as well as fabrics, yarns,fibers, non-woven materials, natural materials, synthetic materials, andany other textile material.

As used herein, “dishwashing composition” refers to all forms ofcompositions for cleaning dishware, including cutlery, including but notlimited to granular and liquid forms. It is not intended that thepresent invention be limited to any particular type or dishwarecomposition. Indeed, the present invention finds use in cleaningdishware (e.g., dishes, including, but not limited to plates, cups,glasses, bowls, etc.) and cutlery (e.g., utensils, including but notlimited to spoons, knives, forks, serving utensils, etc.) of anymaterial, including but not limited to ceramics, plastics, metals,china, glass, acrylics, etc. The term “dishware” is used herein inreference to both dishes and cutlery.

As used herein, “wash performance” of an enzyme refers to thecontribution of an enzyme to washing that provides additional cleaningperformance to the detergent without the addition of the enzyme to thecomposition. Wash performance is compared under relevant washingconditions.

The term “relevant washing conditions” is used herein to indicate theconditions, particularly washing temperature, time, washing mechanics,sud concentration, type of detergent and water hardness, actually usedin households in a detergent market segment.

The term “improved wash performance” is used to indicate that a betterend result is obtained in stain removal from items washed (e.g., fabricsor dishware and/or cutlery) under relevant washing conditions, or thatless enzyme, on weight basis, is needed to obtain the same end resultrelative to another enzyme.

The term “retained wash performance” is used to indicate that the washperformance of an enzyme, on weight basis, is at least 80% relative toanother enzyme under relevant washing conditions.

Wash performance of enzymes is conveniently measured by their ability toremove certain representative stains under appropriate test conditions.In these test systems, other relevant factors, such as detergentcomposition, sud concentration, water hardness, washing mechanics, time,pH, and/or temperature, can be controlled in such a way that conditionstypical for household application in a certain market segment areimitated.

As used herein, the term “bleaching” refers to the treatment of amaterial (e.g., fabric, laundry, pulp, etc.) or surface for a sufficientlength of time and under appropriate pH and temperature conditions toeffect a brightening (i.e., whitening) and/or cleaning of the material.Examples of chemicals suitable for bleaching include but are not limitedto ClO₂, H₂O₂, peracids, NO₂, etc.

As used herein, the term “disinfecting” refers to the removal ofcontaminants from the surfaces, as well as the inhibition or killing ofmicrobes on the surfaces of items. It is not intended that the presentinvention be limited to any particular surface, item, or contaminant(s)or microbes to be removed.

As used herein, the term “perhydrolase” refers to an enzyme that iscapable of catalyzing a reaction that results in the formation ofsufficiently high amounts of peracid suitable for applications such ascleaning, bleaching, and disinfecting. In some preferred embodiments,the perhydrolases encompassed by the present invention include the M.smegmatis perhydrolase, variants and/or homologs thereof as described inPCT/US05/056782. However, it is not intended that the present inventionbe limited to this specific M. smegmatis perhydrolase, specific variantsof this perhydrolase, nor specific homologs of this perhydrolase.

Polyol Oxidases

Polyols, or sugar alcohols, are polyhydric alcohols produced byhydrogenation or fermentation of different carbohydrates. Chemically,polyols are derived from mono- and disaccharides. Most polyols occurnaturally in a variety of food products like vegetables, fruits andmushrooms. They are also regularly presented in fermented foods likewine or soy sauces. Polyols are therefore a normal constituent of thehuman diet. Polyols comprise a variety of sugar alcohols such assorbitol, galactitol, lactitol, xylitol, and mannitol, or alcohols suchas glycerol, propylene glycol and in addition to being constituents ofhuman diet they are also commonly used in personal care products, foodapplications, surfactants, vitamins, plastics and in enzyme productformulations. Polyols are converted to their corresponding sugars andperoxide (H2O2) by polyol oxidase enzymes. Thus, polyol oxidases areattractive biobleaching agents for use in detergents, personal care(e.g., toothpastes, cosmetics, etc.), and other products thatincorporate polyol-containing enzyme product formulations.

The invention encompasses polyol oxidases that can be derived orisolated from Streptomyces, Acidothermus, Arthrobacter, Brevibacterium,Frankia, Nocardia, Janibacter, Marinobacter, Burkholderia, Paracoccus,Chromabacterium, Thermobifida, Xanthomonas, Pseudomonas, Corynebacteriumand Bacillus. In some embodiments, the invention encompasses pololoxidases of the class E.E.1.1.3. In some preferred embodiments, theinvention encompasses polyol oxidases including sorbitol oxidase (SOx),glycerol oxidase (GLOx), xylitol oxidase (XOx), and alditol oxidase(ALOx). In some preferred embodiments, the invention encompassescombining a polyol oxidase e.g. sorbitol oxidase with a second oxidasesuch as glucose oxidase, hexose oxidase, pyranose oxidase,glucooligosaccharide oxidase.

Sorbitol oxidase (“SOx”) is an enzyme that catalyzes conversion ofsorbitol to glucose and hydrogen peroxide. Sorbitol oxidases are knownand used in various settings, including diagnostic methods (See e.g.,Oda and Hiraga, Ann. NY Acad. Sci., 864:454-457 [1998]; and Yamashita etal J. Biosci. Bioengin., 89:350-360 [2000]). Sorbitol (D-glucitol,C₆H₁₄O₆, MW 182.2, CAS 50-70-4) is commonly used in personal careproducts, food applications, surfactants, vitamins, plastics and inenzyme product formulations. In preferred embodiments of the invention,SOx is used in cleaning, bleaching and/or disinfecting compositions.

In some particularly preferred embodiments, the sorbitol oxidase of thepresent invention has a higher specific activity, (or V_(max)/K_(km),ratio) on sorbitol substrate, as compared to an alternative substrateunder standard assay conditions (e.g, in the in vitro assay providedbelow) and/or using an in situ in an application media, conducted asknown in the art. In some preferred alternative embodiments, thealternative substrate is xylitol. In some particularly preferredembodiments, the sorbitol oxidase of the present invention has nosignificant activity on the corresponding sugar product, such asglucose, xylose, galactose (3.5%), mannose (1%) or arabinose.

In some preferred embodiments, the SOx is an oxidoreductase that usescovalently bound FAD as a cofactor for oxidation of sorbitol to glucose.This enzyme offers a unique opportunity for its potential use as abiobleach agent on its own, as well as used in combination with otheroxidases such as carbohydrate oxidases e.g. glucose oxidase and/orhexose oxidase (See e.g., WO 96/39851), pyranose oxidase (See e.g.,WO04/100669), glucooligosaccharide oxidase and M. nivale carbohydrateoxidase (See e.g., WO99/31990). An advantage of the use of suchcombinations is due to the fact that SOx converts sorbitol to glucose,which can then be converted to gluconate by glucose oxidase and/orhexose oxidase and/or carbohydrate oxidases (e.g., Michrodochium nivalecarbohydrate oxidase) and/or glucooligosaccharide oxidase (e.g.,Acremonium strictum glucooligosacharide oxidase), and/or pyranoseoxidase thus generating two moles of hydrogen peroxide per mole ofsorbitol, as illustrated below.

D-Sorbitol+O₂→D-Glucose+H₂O₂

D-Glucose+O₂→D-Gluconate+H₂O₂

In addition, the preferred sorbitol oxidase provided by the presentinvention produces glucose, an aldehyde product that can be furtheroxidized to gluconic acid, a carboxylic acid product using otheroxidases such as hexose oxidase, releasing another molecule of hydrogenperoxide from starting substrate sorbitol. Similarly oxidation ofpolyols such as xylitol, arabitol, mannitol, ribitol, inositol, bysorbitol oxidase, xylitol oxidase, mannitol oxidase with the assistanceof atmospheric oxygen with formation of, xylose, arabinose, mannose,ribose, respectively as secondary substrate for further oxidation byother relevant oxidases such as hexose oxidase, xylose oxidase, pyranoseoxidase, arabinose oxidase, mannose oxidase, and ribose oxidase isfeasible.

Thus, in some embodiments, a polyol oxidase e.g. SOx can be used alone,while in other embodiments, SOx can be used in combination with at leastanother oxidase that catalyzes the conversion of the product of the SOxreaction. For example, SOx can be used in combination with any one ormultiple sugar oxidase enzymes of the class EC1.1.3. For example, SOxcan be used in combination with at least one additional sugar oxidaseincluding but not limited to GOx and/or HOx. In other embodiments, SOxcan be used in combination with other oxidases that catalyze theconversion of polyol substrates that are not the products of the SOxreaction.

In yet other embodiments, SOx can be used alone, while in otherembodiments, SOx can be used in combination with other oxidase enzymesand/or enzymes that convert the H₂O₂ product of oxidase reactions toperacid. For example, SOx can be used in combination with a secondoxidase e.g. glucose oxidase, and at least one perhydrolase enzyme.Examples of perhydrolase enzymes that can be used in combination withthe POx enzymes described herein are described in PCT/05/056782. Thefollowing equation provides an example of a coupled system that findsuse with the present invention.

${Sorbitol} + {O_{2}\overset{{Sorbitol}\mspace{14mu} {oxidase}}{}{glucose}} + {H_{2}O_{2}}\mspace{79mu} + {Glucose} + {H_{2}{O\overset{{Glucose}\mspace{14mu} {oxidase}}{}{gluconic}}\mspace{14mu} {acid}} + {H_{2}O_{2}}\mspace{79mu} + {H_{2}O_{2}} + {{ester}\mspace{14mu} {{substrate}\overset{Perhydrolase}{}{alcohol}}} + {peracid}$

POx enzymes can also be used in combination with bleach activators suchas TAED, NOBS etc., which can be activated by H₂O₂.

Other POx enzymes that find use in the present invention include xylitoloxidase, glycerol oxidase and alditol oxidase.

Xylitol oxidase (“XOx”) is a monomeric oxidase containing one moleculeof FAD per molecule of protein, and it can be derived from Streptomycessp. IKD472. The enzyme catalyzes the conversion of xylitol to glucoseand H2O2, and can also oxidize D-sorbitol (Yamashita et al., J. Biosci.Bioeng. 89 (2000) 350-360). Thus, the invention encompasses cleaning,disinfecting and/or antimicorbial compositions that comprise XOx. Insome embodiments, the compositions of the invention comprise XOx incombination with at least one other sugar oxidase. In other embodiments,compositions of the invention comprise XOx in combination with at leastone other sugar oxidase and/or a perhydrolase.

In some embodiments, the present invention encompasses a xylitol oxidasethat has a higher specific activity, (or V_(max)/K_(km) ratio) onxylitol substrate, as compared to sorbitol under standard assayconditions (e.g., in the in vitro assay provided herein) and/or in an insitu in an application media, conducted as known in the art. In somepreferred alternative embodiments, the alternative substrate issorbitol.

Glycerol oxidase (GLOX) is an enzyme found in the genera Penicillium andBotrytis (See e.g., Lin et al Enz. Micro. Technol., 18:383-387 [1996];and Uwajima et al, Agric. Biol. Chem., 44:399-406 [1989]). This enzymecatalyzes the conversion of glycerol and oxygen to glyceraldehyde andhydrogen peroxide as shown below.

CH₂OH—CHOH—CH₂OH+O₂→CH₂OH—CHOH—CHO+H₂O₂

Glycerol (glycerin, C₃H₈O₃, MW 92.09, CAS 56-81-5) is a commonly used inenzyme product formulations, soap and detergent formulations, food andbeverages, pharmaceuticals and is widely used in cosmetics and personalcare applications. Thus, glycerol oxidase provides an attractivebiobleaching agent for use in detergents that incorporate theseglycerol-containing enzyme product formulations. Thus, the inventionencompasses cleaning, disinfecting and/or antimicorbial compositionsthat comprise GLOx. In some embodiments, the compositions of theinvention comprise GLOx in combination with at least one other polyoloxidase. In other embodiments, compositions of the invention compriseGLOx in combination with at least one other polyol oxidase and/or aperhydrolase. In other embodiments, GLOx can be combined with a bleachactivator.

In some embodiments, the invention encompasses hexose oxidases (“HOx”)(Sullivan, et al., Biochim. Biophys. Acta 309 (1973) 11-22; Bean et al.,J. Biol. Chem. 218 (1956); Bean et al., J. Biol. Chem. 236 (1961)),which catalyze the conversion of D-glucose to D-glucono-1,5-lactone andH2O2. Hexose oxidase also utilizes other substrates includingD-galactose, D-mannose, maltose, lactose and cellobiose, Thus, theinvention encompasses cleaning, disinfecting and/or antimicorbialcompositions that comprise HOx. In some embodiments, the compositions ofthe invention comprise HOx in combination with at least one other polyoloxidase. In other embodiments, compositions of the invention compriseHOx in combination with at least one other polyol oxidase and/or aperhydrolase.

In some embodiments, the compositions of the invention comprise glucoseoxidase (GOx), which catalyzes the conversion of glucose toD-glucono-1,5-lactone+H2O2. Thus, GOx can utilize the product of polyoloxidase reactions as a substrate to convert glucose to the correspondinglactone and generate a second molecule of H2O2. Thus, the inventionencompasses cleaning, disinfecting and/or antimicorbial compositionsthat comprise GOx. In some embodiments, the compositions of theinvention comprise GOx in combination with at least one other polyoloxidase. In other embodiments, compositions of the invention compriseGOx in combination with at least one other polyol oxidase and/or aperhydrolase. In other embodiments, compositions of the inventioncomprise GOx in combination with at least one other POx and/or a bleachactivator.

As indicated above, key components to peracid production by enzymaticperhydrolysis are enzyme, ester substrate, and hydrogen peroxide. In thepresent invention, hydrogen peroxide is generated in a coupled enzymesystem that includes at least one POx to provide the H₂O₂ substrate forthe perhydrolase. In some embodiments, the POx oxidase that can be usedin cleaning compositions in combination with a perhydrolase can be apolyol oxidases of the class E.E.1.1.3. For example, enzymes (e.g.,sorbitol oxidase, glucose oxidase, hexose oxidase, xylitol oxidase,alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acidoxidase, etc.) that can generate hydrogen peroxide also find use withester substrates in combination perhydrolase enzymes to generateperacids. In some preferred embodiments, the invention encompassescleaning compositions comprising a combination of a perhydrolase with asorbitol oxidase (SOx), glycerol oxidase, a glucose oxidase (GOx), axylitol oxidase (XOx), and/or hexose oxidase (XOx). The inventionincludes cleaning compositions that include combinations of one or moreperhydrolases with one/or more POx enzymes. In some embodiments, theinvention cleaning compositions and methods that include a perhydrolasein combination with a sorbitol oxidase and a glucose oxidase. In otherembodiments, the invention provides cleaning compositions and methodsthat encompass a perhydrolase in combination with a sorbitol oxidase anda hexose oxidase. In yet other embodiments, the invention providescleaning compositions and methods that encompass a perhydrolase. In yetother embodiments, the invention provides compositions and methods thatencompass a perhydrolase in combination with xylitol oxidase and apyranose oxidase. In some embodiments, the compositions of the inventioncomprise at least one POx enzyme and a bleach activator. The inventionalso provides for the use of additional sources of H₂O₂ that can be usedwith POx alone or in combination with perhydrolase enzymes. For example,chemical sources of H₂O₂ such as percarbonates and perborates, whichspontaneously decompose to H₂O₂, which are included in some currentwashing powders find use in the present invention. One advantage of themethods of the present invention is that the generation of acid by a POx(e.g., gluconic acid in the above example) reduces the pH of a basicsolution to the pH range in which the peracid is most effective inbleaching (i.e., at or below the pKa). In some preferred embodiments,the ester substrates are selected from one or more of the followingacids: formic acid, acetic acid, propionic acid, butyric acid, valericacid, caproic acid, caprylic acid, nonanoic acid, decanoic acid,dodecanoic acid, myristic acid, palmitic acid, stearic acid, and oleicacid. Importantly, the present invention provides means for effectivecleaning, bleaching, and disinfecting over broad pH and temperatureranges. In some embodiments, the compositions of the invention provideeffective cleaning, bleaching, and disinfecting at pH between 5.5 and10.5. The preferred temperatures at which the composition of theinvention provide effective cleaning, bleaching, and disinfecting rangebetween 10 and 70° C. Other POx enzymes that find use in the presentinvention are described in PCT Applications DK2006/000590 andDK2006/000591, which are herein incorporated by reference in theirentirety.

The present invention encompasses compositions and methods forexpressing polyol oxidases in microorganisms. Polyol oxidasesencompassed by the invention include oxidases that can react with asample containing at least one polyol selected from the group consistingof D-sorbitol, D-mannitol, D-xylitol, and D-arabitol to produce hydrogenperoxide and D-glucose, D-mannose, D-xylose, or D-arabinose,respectively. In some embodiments, the invention encompasses polyoloxidases that are sorbitol oxidases. In other embodiments, the inventionencompasses polyol oxidases that are xylitol oxidases.

In some embodiments, the polyol enzymes of the invention havespecificity for D-sorbitol and D-xylitol and thus possess both sorbitoloxidase and xylitol oxidase activity. It is understood that the polyoloxidses encompassed by the invention are not limited to oxidases thatutilize any one particular substrate but can utilize any one or acombination of substrates including D-sorbitol, D-xylitol, D-mannitol,D-ribitol, myo-inositol, glycerol, 1,3 propanediol and 1,2 propanediol.

The polyol oxidases of the present invention can be isolated from anumber of microorganisms including but not limited to Streptomyces sp.,Bacillus sp., Acidothermus sp., Arthobacter sp., and marineAcinobacteium sp. In some preferred embodiments, the Streptomyces sp.include Streptomyces strain H-7775, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces lividans and Streptomyces speciesIKD472. In other preferred embodiments, the Acidothermus sp. isAcidothermus cellulolyticus 11B (Q2E2H5). In some embodiments, theArthobacter sp. is Arthrobacter sp. FB24 (Q4NJLO). In some embodiments,the marine Actinobacterium is Actinobacterium PHSC20C1.

In some embodiments, the invention provides for isolated recombinantpolynucleotides that encode a POx. In some embodiments, the recombinantpolynucleotides comprise a sequence encoding the mature form of the POxthat is a wild-type sequence. In other embodiments, the recombinantpolynucleotides comprise a sequence encoding the mature form of the POxthat is a variant, homolog or fragment of the wild-type sequence. Insome preferred embodiments, the polynucleotide sequence is the wild-typesequence of the SOx of Streptomyces H-7775 (e.g. SEQ ID NO:2). In otherembodiments, the polynucleotide sequence encoding the POx is thewild-type sequence of the SOx from Streptomyces lividans (e.g. SEQ IDNO:4). In some embodiments, the polynucleotides encoding the POx enzymesof the invention are synthetic polynucleoitdes that encode the aminoacid sequences of the polyol oxidases derived from the Streptomyces sp.,Bacillus sp., Acidothermus sp., Arthobacter sp., and marineAcinobacterium sp. In some preferred embodiments, the syntheticpolynucleotides encode the POx enzymes isolated from Streptomyces sp.including Streptomyces strain H-7775 (e.g. SEQ ID NO:2), Streptomycesavermitilis (e.g. SEQ ID NO:8), Streptomyces coelicolor (e.g. SEQ IDNO:9) and Streptomyces species IKD472 (e.g. SEQ ID NO:10). In otherpreferred embodiments, the synthetic polynucleotides encode the POxenzymes isolated from Acidothermus cellulolyticus 11B (Q2E2H5; SEQ IDNO:6). In some other embodiments, the synthetic polynucleotides encodethe POx enzymes isolated from Arthrobacter sp. FB24 (Q4NJLO; SEQ IDNO:12). In yet other embodiments, the synthetic polynucleotides encodethe POx enzymes isolated from Actinobacterium PHSC20C1 (e.g. SEQ IDNO:13). Other POx sequences that find use in the present inventioninclude but are not limited to polynucleotides that encode the POx ofAcidothermus cellulolyticus (ZP_(—)01136416: e.g. SEQ ID NO:5),Streptomyces avermitilis (NP_(—)823266; e.g. SEQ ID NO:7), Arthrobactersp FB24 (ZP_(—)00411614; e.g. SEQ ID NO:11), and Streptomyces sp. IKD472(Q9KX73; e.g. SEQ ID NO:14). In some embodiments, the POxpolynucleotides encode a POx enzyme that has greater SOx activity thanXOx activity. In other embodiments, the POx polynucleotides encode a POxenzyme that has greater XOx activity than SOx activity when utilizingthe same polyol substrate.

The invention also encompasses polynucleotide sequences, whetherwild-type or synthetic that are derived from genes homologous to thoseof Streptomyces H-7775 and/or Streptomyces lividans. For example, theinvention provides for synthetic polynucleotide sequences e.g. 1, 3, 17,20, 29, 30, 34, and 44 which comprise sequences encoding polyoloxidases. As indicated above, “homologous genes” are genes thatcorrespond to each other and which are identical or very similar to eachother, yet are obtained from different species. The term encompassesgenes that are separated by speciation (i.e., the development of newspecies) (e.g., orthologous genes), as well as genes that have beenseparated by genetic duplication (e.g., paralogous genes). Homologousgenes encode for homologous proteins.

During the development of the present invention, sorbitol oxidases wereisolated from recombinant Streptomyces lividans strains expressing theputative sorbitol oxidase gene from S. lividans and also the known SOxgene from Streptomyces sp. H-7775 (See, Hiraga et al., Biosci. Biotech.Biochem., 61:1699-1704 [1997]). The SOx gene from S. lividans wasidentified based on the sequence derived from the Streptomycescoelicolor SCO6147 annotated as a putative xylitol oxidase gene. Twodifferent synthetic genes encoding the Streptomyces sp. H-7775 sorbitoloxidase gene were used for intracellular expression in E. coli strainBL21(DE3)pLysS and extracellular expression in S. lividans g3s3, whichis a derivative of the TK23 strain in which the cyc2 gene was deleted(Boremann et al., J. Bacteriol 178:1216-1218 (1996)). The prostheticgroup is a covalently bound FAD (1 mol of FAD to 1 mol of SOx). Thus, itis a flavoprotein, with typical absorption maxima at 276, 358, and 455nm. However, in other embodiments, the enzyme exhibits a second maximum,not at 358 but at 345, indicative of a histidine-flavin linkage. Thus,it is not intended that the present invention be limited to anyparticular mechanism and/or embodiment. Flavin is functionally involvedin oxidation of sorbitol as observed by desired changes in UV-VISspectra. FAD is very tightly bound with the protein and thus offers astable enzyme for laundry and personal care (e.g., oral care)applications.

The SOx gene from Streptomyces species H-7775 (SEQ ID NO:1) has beendescribed (See, Genbank accession number AB000519). Applicants haveidentified several homolgs of the SOx from S. H-7775 using a BLASTsearch of the NCBI database. These include but are not limited to axylitol oxidase from Streptomyces avermitilis (BAC69801; Q82LCO; SEQ IDNO:8), a xylitol oxidase from Streptomyces coelicolor (Q9ZBU1; SEQ IDNO:9), a xylitol oxidase from Streptomyces sp. IKD472 (Q9KX73; SEQ IDNO:10), a putative xylitol oxidase from Acidothermus cellulolyticus 11B(Q2E2H5; SEQ ID NO:6), and a FAD-linked oxidase from Arthrobacter spFB24 (ZP00411614; SEQ ID NO:11), a FAD-linked oxidase from Arthrobactersp FB24 (YP_(—)833485; Q4NJLO; SEQ ID NO: 12), a putative xylitoloxidase from marine Actinobacterium PHSC20C1 (GenBank Accession no.ZP_(—)01129132) and a xylitol oxidase from Streptomyces sp. IKD472(GenBank Accession no. Q9KX73; SEQ ID NO:14). As discussed above, insome embodiments, the invention encompasses polynucleotide sequencesthat encode POx that are homologous to that of S. H7775 (SEQ ID NO:2).Thus, the invention encompasses POx enzyme encodes by polynucleotidehomologs of SEQ ID NO: 2.

The sorbitol oxidase gene from Streptomyces species H-7775 (Genbankaccession number AB000519) comprises a 1260 bp open reading frame (ORF)encoding a protein having 420 amino acids with theoretical MW of 45,158Daltons. The enzyme is stable for 24 hours at 30° C., between pH 7.5-10with an optimum temperature of 50° C. at pH 7.5. It is also heat stableup to 55° C.

The nearest homolog identified for this enzyme is xylitol oxidase (with55% homology). SOx is an efficient enzyme for multiple applications,including detergents, fabric care, home care, oral care (e.g., dentalwhitening, antimicrobial and/or cleaning), personal care, textileprocessing, food processing and industrial cleaning. In addition, insome embodiments, SOx can catalyze other substrates. Thus, this enzymeuses wide spectrum of substrates, providing flexibility in substrateusage in various applications. It is noted that many of these substratesare present in typical detergent formulations, or can be added to them.The synthetic gene sequence (neutral codons) and the corresponding aminoacid sequence of sorbitol oxidase from Streptomyces sp. H-7775 areknown. These sequences are set forth in SEQ ID NOS:1 and 2,respectively, as shown below. It is noted that the amino acid sequenceprovided by P97011 (Streptomyces sp.) has been reported to be the sameas that of Streptomyces sp. H-7775. SEQ ID NO. 1 is the synthetic genecloned in an E. coli expression vector pET24a and expressed in E. colistrain BL21(DE3)pLysS.

Thus, in some embodiments, the invention provides for polynucleotidesencoding a wild-type SOx derived from Streptomyces H-7775 (SEQ ID NO:1)

(SEQ ID NO: 1) GGTACCCATA TGACCCCTGC TGAAAAAAAC TGGGCCGGCA ATATCACTTTCGGTGCAAAG AGACTTTGCG TTCCACGTTC TGTCAGAGAG CTGCGCGAAA CAGTTGCTGCCAGTGGAGCA GTGAGACCTT TGGGAACGCG GCACTCCTTT AACACTGTCG CTGACACCTCAGGTGATCAT GTTTCTTTGG CCGGTCTCCC GAGAGTCGTT GACATTGATG TGCCAGGCAGGGCTGTTAGC CTGTCGGCAG GACTTAGATT CGGTGAGTTT GCTGCCGAAT TGCATGCTCGAGGTCTCGCC CTGGCAAATC TGGGCTCACT TCCCCACATT TCTGTCGCTG GGGCCGTGGCAACCGGCACA CATGGAAGTG GAGTGGGTAA CCGTTCCTTG GCCGGTGCTG TCAGAGCACTGAGCCTCGTT ACTGCTGATG GCGAGACACG CACCCTTAGG CGTACTGACG AAGATTTTGCCGGGGCTGTC GTGTCTCTGG GCGCATTGGG AGTTGTGACG TCGCTTGAGT TGGACCTCGTTCCTGCCTTC GAAGTCAGAC AGTGGGTGTA CGAGGATCTG CCAGAAGCTA CACTTGCCGCCAGATTTGAC GAGGTTATGT CCGCTGCATA TAGCGTCAGT GTGTTCACGG ATTGGAGACCGGGTCCTGTT GGACAAGTCT GGCTCAAACA ACGAGTTGGC GACGAAGGGG CTAGATCAGTATGCCCGCA GAGTGGCTGG GTGCCAGATT GGCTGATGGA CCACGTCACC CTGTTCCGGGATGCCAGCC GGTAATTGTA CTGCACAGCA AGGCGTTCCG GGCCCTTGGC ATGAAAGACTGCCCCACTTC CGCATGGAAT TTACCCCATC CAACGGTGAC GAGTTGCAGT CGGAGTATTTTGTCGCTAGG GCTGATGCCG TTGCCGCCTA CGAAGCTCTT GCACGCCTCC GCGACAGAATCGCACCTGTC CTGCAAGTGT CTGAGTTGCG TACAGTCGCT GCCGACGATC TGTGGCTTTCACCGGCTCAT GGAAGAGATA GCGTGGCCTT CCACTTTACC TGGGTTCCAG ACGCTGCCGCAGTCGCTCCT GTGGCCGGTG CAATTGAGGA AGCTCTCGCC CCCTTTGGCG CAAGACCGCATGGGGGAAG GTTTTCTCTA CTGCTCCCGA GGTCCTGCGA ACGTTGTACC CACGCTATGCCGACTTTGAG GAACTTGTGG GACGTCACGA TCCTGAAGGC ACCTTCAGGA ACGCCTTTCTCGATCGCTAC TTCCGGCGTT AATAAGGATC CGAGCTC (GENBANK ACCESSION NO.BAA19135; SEQ ID NO: 2)MTPAEKNWAGNITFGAKRLCVPRSVRELRETVAASGAVRPLGTRHSFNTVADTSGDHVSLAGLPRVVDIDVPGRAVSLSAGLRFGEFAAELHARGLALANLGSLPHISVAGAVATGTHGSGVGNRSLAGAVRALSLVTADGETRTLRRTDEDFAGAVVSLGALGVVTSLELDLVPAFEVRQWVYEDLPEATLAARFDEVMSAAYSVSVFTDWRPGPVGQVWLKQRVGDEGARSVMPAEWLGARLADGPRHPVPGMPAGNCTAQQGVPGPWHERLPHFRMEFTPSNGDELQSEYFVARADAVAAYEALARLRDRIAPVLQVSELRTVAADDLWLSPAHGRDSVAFHFTWVPDAAAVAPVAGAIEEALAPFGARPHWGKVFSTAPEVLRTLYPRYADFEELVGRHDPEGT FRNAFLDRYFRR

The nucleic and amino acid sequences of the putative sorbitol oxidasesin Streptomyces lividans and Streptomyces coelicolor are provided below(SEQ ID NOS:3 and 4, respectively). The addition of the NcoI cloningsite at the start methionine resulted in amino acid change from serineto glycine of the second amino acid residue at the N-terminus. This isthe gene cloned and expressed as an intracellular protein inStreptomyces lividans g3s3 showing both sorbitol and xylitol oxidaseactivities, as described herein.

(SEQ ID NO: 3) GCCATGGGCG ACATCACGGT CACCAACTGG GCCGGCAACA TCACGTACACGGCGAAGGAA CTGCTGCGGC CGCACTCCCT GGACGCGCTG CGGGCCCTGG TGGCGGACAGCGCCAGGGTG CGGGTGCTGG GCAGCGGGCA CTCCTTCAAC GAGATCGCCG AGCCGGGCGACGGGGGTGTC CTGCTGTCGC TGGCGGGCCT GCCGTCCGTG GTGGACGTGG ACACGGCGGCCCGTACGGTG CGGGTCGGCG GCGGTGTGCG GTACGCGGAG CTGGCCCGGG TGGTGCACGCGCGGGGCCTG GCGCTGCCGA ACATGGCCTC GCTGCCGCAC ATCTCGGTCG CCGGGTCGGTGGCCACCGGC ACCCACGGTT CGGGGGTGGG CAACGGTTCG CTGGCCTCGG TGGTGCGCGAGGTGGAGCTG GTCACCGCGG ACGGTTCGAC CGTGGTGATC GCGCGGGGCG ACGAGCGGTTCGGCGGGGCG GTGACCTCGC TCGGCGCGCT GGGCGTGGTG ACGTCGCTCA CACTCGACCTGGAGCCGGCG TACGAGATGG AACAGCACGT CTTCACCGAG CTGCCGCTGG CCGGGTTGGACCCGGCGACG TTCGAGACGG TGATGGCGGC GGCGTACAGC GTGAGTCTGT TCACCGACTGGCGGGCGCCC GGTTTCCGGC AGGTGTGGCT GAAGCGGCGC ACCGACCGGC CGCTGGACGGTTTCCCGTAC GCGGCCCCGG CCGCCGAGAA GATGCATCCG GTGCCGGGCA TGCCCGCGGTGAACTGCACG GAGCAGTTCG GGGTGCCGGG GCCCTGGCAC GAGCGGCTGC CGCACTTCCGCGCGGAGTTC ACGCCCAGCA GCGGTGCCGA GTTGCAGTCG GAGTACCTGA TGCCCCGGGAGCACGCCCTG GCCGCCCTGC ACGCGATGGA CGCGATACGG GAGACGCTCG CGCCGGTGCTCCAGACCTGC GAGATCCGCA CGGTCGCCGC CGACGCGCAG TGGCTGAGCC CGGCGTACGGGCGGGACACC GTGGCCGCGC ACTTCACCTG GGTCGAGGAC ACGGCGGCGG TGCTGCCGGTGGTGCGGCGG CTGGAGGAGG CGCTCGTCCC CTTCGCGGCC CGTCCGCACT GGGGGAAGGTGTTCACCGTC CCGGCGGGCG AGCTGCGTGC GCTGTACCCG CGGCTGGCCG ACTTCGGGGCGCTGGCCGGG GCGCTGGACC CGGCGGGGAA GTTCACCAAC GCGTTCGTGC GCGGGGTGCTCGCGGGCTGA GGATCCAT (SEQ ID NO: 4) MGDITVTNWA GNITYTAKEL LRPHSLDALRALVADSARVR VLGSGHSFNE IAEPGDGGVL LSLAGLPSVV DVDTAARTVR VGGGVRYAELARVVHARGLA LPNMASLPHI SVAGSVATGT HGSGVGNGSL ASVVREVELV TADGSTVVIARGDERFGGAV TSLGALGVVT SLTLDLEPAY EMEQHVFTEL PLAGLDPATF ETVMAAAYSVSLFTDWRAPG FRQVWLKRRT DRPLDGFPYA APAAEKMHPV PGMPAVNCTE QFGVPGPWHERLPHFRAEFT PSSGAELQSE YLMPREHALA ALHAMDAIRE TLAPVLQTCE IRTVAADAQWLSPAYGRDTV AAHFTWVEDT AAVLPVVRRL EEALVPFAAR PHWGKVFTVP AGELRALYPRLADFGALAGA LDPAGKFTNA FVRGVLAG

The sequences of other sorbitol oxidases that are homologs of SEQ IDNO:2 or 4 are encompassed by the invention and include those set forthbelow. It is understood that any homolog of the SOx of SEQ ID NOs: 2 or4 that retains sorbitol and/or xylitol oxidase activity is encompassedby the invention.

Acidothermus cellulolyticus (ZP_01136416) (SEQ ID NO: 5) MDGGKRCRDGTPQPPAPSEQ VTPSAAASLR AAYDVEVSAP RLRNWAGNIAFRPRRYVQPRDLDELVEIIRVSDQVRVLGTGHSFNPIADTTGTLISLDHLPREVRVMPGRTAVSAGTRYGDLAFPLHEAG WALANVGSLP HISIAGACAT ATHGSGDRNGCLATAVAGMTGVDGTCRVFHLTAESPEFPGAVVHLGALGAVTEIELVTEPTFTVRQWVYEDAPLDNVFADLDDVTSAAYSVSIFTTWDPPTARQIWLKERVAAGRPDPPA RRWGGRLAERDHNPVPGMPP ENCTPQLGRIGPWHERLPHF RLDVTPSAGDELQSEYFVPRAAAVEAYRALRHIGSRIAPVLQISEIRTVAADELWLSPAYHRPSVAFHFTWIADEEAVRPVVSEVERALAPLQPRPHWGKLFTMDPAVVRAAYPRFDDFV ALAERYDPEG KFQNDFLRRFFAG Acidothermus cellulolyticus (11B Q2E2H5) (SEQ ID NO: 6) MDGGKRCRDGTPQPPAPSEQ VTPSAAASLR AAYDVEVSAP RLRNWAGNIA FRPRRYVQPR DLDELVEIIRVSDQVRVLGT GHSFNPIADT TGTLISLDHL PREVRVMPGR TAVSAGTRYG DLAFPLHEAGWALANVGSLP HISIAGACAT ATHGSGDRNG CLATAVAGMT GVDGTCRVFH LTAESPEFPGAVVHLGALGA VTEIELVTEP TFTVRQWVYE DAPLDNVFAD LDDVTSAAYS VSIFTTWDPPTARQIWLKER VAAGRPDPPA RRWGGRLAER DHNPVPGMPP ENCTPQLGRI GPWHERLPHFRLDVTPSAGD ELQSEYFVPR AAAVEAYRAL RHIGSRIAPV LQISEIRTVA ADELWLSPAYHRPSVAFHFT WIADEEAVRP VVSEVERALA PLQPRPHWGK LFTMDPAVVR AAYPRFDDFVALAERYDPEG KFQNDFLRRF FAG Streptomyces avermitilis (NP_823266) (SEQ IDNO: 7) MTDAGTALTN WAGNITYSAK ELHRPQSLDA LRALVADSAK VRVLGSGHSFNEIAEPGADGVLLSLTALPP SVEVDTAART VRVAGGVRYA ELARVVHGHG LALPNMASLPHISVAGSVATGTHGSGVTNG SLASAVREVE LVTADGSAVR IGRGDDRFDG AVTALGALGVVTALTLDLEPDYRVAQQVFT ELPLAGLDFD AVAASAYSVS LFTGWRTSGF AQVWLKRRTDRPSADFPWAAPATEAMHPVP GMPAVNCTQQ FGVPGPWHER LPHFRAEFTP SSGAELQSEYLLPRPYALDA LHALDAVRET VAPVLQICEV RTVAADAQWL SPAYGRDTVA LHFTWVEDLAAVLPVVRRVEEALDPFDPRP HWGKVFAVPA RVLRGRYPRL GDFRALVDSL DPGGKFTNAFVREVLGSGDRPS Streptomyces avermitilis (Q82LCO) (SEQ ID NO: 8) MTDAGTALTNWAGNITYSAK ELHRPQSLDA LRALVADSAK VRVLGSGHSF NEIAEPGADG VLLSLTALPPSVEVDTAART VRVAGGVRYA ELARVVHGHG LALPNMASLP HISVAGSVATGTHGSGVTNGSLASAVREVE LVTADGSAVR IGRGDDRFDG AVTALGALGV VTALTLDLEP DYRVAQQVFTELPLAGLDFD AVAASAYSVS LFTGWRTSGF AQVWLKRRTD RPSADFPWAA PATEAMHPVPGMPAVNCTQQ FGVPGPWHER LPHFRAEFTP SSGAELQSEY LLPRPYALDA LHALDAVRETVAPVLQICEV RTVAADAQWL SPAYGRDTVA LHFTWVEDLA AVLPVVRRVE EALDPFDPRPHWGKVFAVPA RVLRGRYPRL GDFRALVDSL DPGGKFTNAF VREVLGSGDR PS Streptomycescoelicolor (Q9ZBU1) (SEQ ID NO: 9) MSDITVTNWA GNITYTAKEL LRPHSLDALRALVADSARVR VLGSGHSFNE IAEPGDGGVLLSLAGLPSVV DVDTAARTVR VGGGVRYAELARVVHARGLA LPNMASLPHI SVAGSVATGTHGSGVGNGSL ASVVREVELV TADGSTVVIARGDERFGGAV TSLGALGVVT SLTLDLEPAYEMEQHVFTEL PLAGLDPATF ETVMAAAYSVSLFTDWRAPG FRQVWLKRRT DRPLDGFPYAAPAAEKMHPV PGMPAVNCTE QFGVPGPWHERLPHFRAEFT PSSGAELQSE YLMPREHALAALHAMDAIRE TLAPVLQTCE IRTVAADAQWLSPAYGRDTV AAHFTWVEDT AAVLPVVRRLEEALVPFAAR PHWGKVFTVP AGELRALYPRLADFGALAGA LDPAGKFTNA FVRGVLAG Streptomyces sp. IKD472/FERM P-14339(Q9KX73) (SEQ ID NO: 10) MSTAVTNWAG NITYTAKEVH RPATAEELAD VVARSAWGACAGAAGHSFNE IADPGPDGVLLRLDALPAETDVDTTARTVRVGGGVRYAELARVVHAHGLALPNMASLPHISVAGSVATGTHGSGVTNGPLAAPVREVELVTADGSQVRIAPGERRFGGAVTSLGALGVVTALTLDLEPAFEVGQHLFTELPLRGLDFETVAAAGYSVSLFTDWREPGFRQVWLKRRTDQELPDFPWARPATVALHPVPGMPAENCTQQFGVPGPWHERLP HFRAEFTPSS GAELQSEYLLPRAHALDALDAVDRIRDTVA PVLQTCEVRT VAPDEQWLGP SHGRDTVALH FTWVKDTEAVLPVVRRLEEALDAFDPRPHW GKVFTTSAAA LRARYPRLAD FRALARELDP SGKFTNTFLR DLLDGArthrobacter sp. FB24 (ZP_00411614) (SEQ ID NO: 11) MRTVSELPGLSGSTGAGSSA PELNWAGNYR YTAASIHRPR TLEEVQEVVAGASKIRALGSRHSFNAIADSPGSLVSLEDL DPGIRIDAAT RTVTVSGGTR YGTLAEQLES AGFALSNLASLPHISVAGAIATATHGSGDA NGNLATSVAA LELVAADGTV HRLNRGSSPG FDGAVVGLGALGVVTKVTLDIEPTFTVRQD VFEALPWDTV LGNFDAVTSS AYSVSLFTDW SGDDVAQAWL KSRLSGSAASSDAGSTLAGE AFAAGTFFGG TRAGVARHPL PGVSAENCTE QLGVPGSWSERLAHFRMAFTPSSGEELQSE FFVRREHAVA AIGELRALSD RITPLLLVSE IRTVAADKLWLSTAYGQDSVGFHFTWKQRQ DEVEKVLPVM EEALAPFNAR PHWGKLFHAG ADAVAELYPRFSDFKDLAERMDPEQKFRNE FLARKVFGN Arthrobacter sp. FB 24 (Q4NJLO; GenBank Accessionno. YP_833485) (SEQ ID NO: 12) MRTVSELPGL SGSTGAGSSA PELNWAGNYRYTAASIHRPR TLEEVQEVVA GASKIRALGS RHSFNAIADS PGSLVSLEDL DPGIRIDAATRTVTVSGGTR YGTLAEQLES AGFALSNLAS LPHISVAGAI ATATHGSGDA NGNLATSVAALELVAADGTV HRLNRGSSPG FDGAVVGLGA LGVVTKVTLD IEPTFTVRQD VFEALPWDTVLGNFDAVTSS AYSVSLFTDW SGDDVAQAWL KSRLSGSAAS SDAGSTLAGE AFAAGTFFGGTRAGVARHPL PGVSAENCTE QLGVPGSWSE RLAHFRMAFT PSSGEELQSE FFVRREHAVAAIGELRALSD RITPLLLVSE IRTVAADKLW LSTAYGQDSV GFHFTWKQRQ DEVEKVLPVMEEALAPFNAR PHWGKLFHAG ADAVAELYPR FSDFKDLAER MDPEQKFRNE FLARKVFGN MarineActinobacterium PHSC20C1 (ZP_01129132) (SEQ ID NO: 13) MLTNQTNWAGNLTYNAKAIM QPTNVDELQE LVARLPRVRA LGTRHSFTDI ADTPGTLMSLANMPPNIHIDTTAMTASVTG GTSYGLLMSE LQSNGFALHN TGSLPHISVA GATATATHGSGDGNGILSTAIAALDVVTAD GSLVTVDRAS DHLPALAVGL GAFGVIARVT LDIEPTYRVRQDVYRFAPWETVLEQLDDIM ASAYSVSLLA DFGSPTVAQI WLKTRLGVGD DPEVAPTLFGGIWYDDSDELAPQNVNQRAS IPGPWSERMP HFRLDGEPSN GGDELQSEYY VRREHGVQALEALRGLGAQISPHLLISEIR TAAADSLWMS PAYGQDVLCI GFTWAKHPAE VTALLPEIEATLAPFAPRQHWGKLFSFSRD IIAERFPRVA DFTELRDQYD PQRKFWNPFL ERTLGAP

In additional embodiments, xylitol oxidases find use in the presentinvention. The following sequence is a xylitol oxidase.

Streptomyces sp.IKD472 (Q9KX73) (SEQ ID NO: 14) MSTAVTNWAG NITYTAKEVHRPATAEELAD VVARSAWGAC AGAAGHSFNE IADPGPDGVL LRLDALPAET DVDTTARTVRVGGGVRYAEL ARVVHAHGLA LPNMASLPHI SVAGSVATGT HGSGVTNGPL AAPVREVELVTADGSQVRIA PGERRFGGAV TSLGALGVVT ALTLDLEPAF EVGQHLFTEL PLRGLDFETVAAAGYSVSLF TDWREPGFRQ VWLKRRTDQE LPDFPWARPA TVALHPVPGM PAENCTQQFGVPGPWHERLP HFRAEFTPSS GAELQSEYLL PRAHALDALD AVDRIRDTVA PVLQTCEVRTVAPDEQWLGP SHGRDTVALH FTWVKDTEAV LPVVRRLEEA LDAFDPRPHW GKVFTTSAAALRARYPRLAD FRALARELDP SGKFTNTFLR DLLDG

In some embodiments, the POx enzymes of the invention are encoded bychimeric polynucleotides that comprise a polynucleotide that encodes themature form of the POx enzyme that is operably linked to apolynucleotide that encodes a signal peptide. In some embodiments, thechimeric polynucleotide is a synthetic chimeric polynucleotide. In otherembodiments, the chimeric polynucleotide comprises a wild-type sequencethat encodes the signal peptide and a synthetic sequence that encodesthe mature form of the POx enzyme.

In prokaryotes two pathways for protein translocation across thecytoplasmic membrane have been recognized. In most bacteria the generalsecretory (Sec) pathway is the best-characterized route for proteinexport. Proteins exported by this pathway are translocated across themembrane in an unfolded state through a membrane-embedded translocon towhich they are targeted by cleavable N-terminal signal peptides (Mori etal., (2001) Trends in Microbiology 9:494-500). More recently a secondgeneral export pathway has been described, which is designated thetwin-arginine translocation (Tat) pathway and reference is made to US2002/0110860; WO 03/079007; Berks, B. C. (1996) Mol. Microbiol.22:393-404 and Tjalsma et al., (2000) Microbiol. & Molecul. Bio. Reviews64:515-547.

The choice of signal sequence largely depends on the host cell used. Asnoted above, in certain embodiments, a Streptomyces and/or a Bacillushost cell is employed, the signal sequence may be any sequence of aminoacids that is capable of directing the fusion protein into the TAT orSEC pathway of the Streptomyces and/or Bacillus host cell.

In some embodiments, signal sequences that may be employed include thesignal sequences of proteins that are secreted from wild-type Bacilluscells. Such signal sequences include the signal sequences encoded byα-amylase, protease, (e.g., aprE or subtilisin E), or β-lactamase genes.Exemplary signal sequences include, but are not limited to, the signalsequences encoded by an α-amylase gene, an subtilisin gene, aβ-lactamase gene, a neutral protease gene (e.g., nprT, nprS, nprM), or aprsA gene from any suitable Bacillus species, including, but not limitedto B. stearothermophilus, B. licheniformis, B. lentus, B. subtilis, andB. amyloliquefaciens. In some embodiments, the signal sequence isencoded by the aprE gene of B. subtilis (See e.g., Appl. Microbiol.Biotechnol., 62:369-73 [2003]). Further signal peptides find use in thepresent invention (See e.g., Simonen and Palva, Micro. Rev., 57:109-137[1993]; etc.). A preferred signal peptide encompassed by the inventionis the signal peptide of the Bacillus circulans cyclomaltodextringlucanotransferase (cgt) precursor (Accession no. P43379; SEQ ID NO:36).

In other embodiments, the signal sequence that may be employed includesis a signal sequences of a protein that is secreted from the wild-typeStreptomyces host cells. Such signal sequences include the signalsequence encoded by the S. coelicolor SCO6772 gene (SEQ ID NO:15), thesignal sequence encoded by the S. coelicolor celA gene (EMBL accessionno. AL939132.1; SEQ ID NO: 27); the signal sequence encoded by the S.coelicolor secreted endoglucanase gene SCO7363 (SEQ ID NO:31), or thesignal sequence encoded by the S. coelicolor possible secreted proteingene SCO0624 (SEQ ID NO: 39). Other signal peptides encoded by theStreptomyces sp. genes are described in WO2007/071996 and in Widdick etal. (Science 103:17927-17932 (2006).

In some embodiments, the POx polynucleotides encode mature POxpolypeptides and/or POx fusion polypeptides that share at least about65% amino acid sequence identity, preferably at least about 70% aminoacid sequence identity, more preferably at least about 75% amino acidsequence identity, still more preferably at least about 80% amino acidsequence identity, more preferably at least about 85% amino acidsequence identity, even more preferably at least about 90% amino acidsequence identity, more preferably at least about 92% amino acidsequence identity, yet more preferably at least about 95% amino acidsequence identity, more preferably at least about 97% amino acidsequence identity, still more preferably at least about 98% amino acidsequence identity, and most preferably up to about 99% amino acidsequence identity with the amino acid sequence of the precursor POxprotein and have comparable or enhanced production activity, as comparedto the precursor polypeptide.

As will be understood by the skilled artisan, due to the degeneracy ofthe genetic code, a variety of POx polynucleotides encode fusion andmature POx proteins. In some other embodiments of the present invention,polynucleotides comprising a nucleotide sequence having at least about70% sequence identity, at least about 75% sequence identity, at leastabout 80% sequence identity, at least about 85% sequence identity, atleast about 90% sequence identity, at least about 92% sequence identity,at least about 95% sequence identity, at least about 97% sequenceidentity, at least about 98% sequence identity and at least about 99%sequence identity to the polynucleotide sequence of SEQ ID NOS:2, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 28, 33, 35, 37, 38, and/or 40, areprovided.

In some embodiments, the percent identity shared by polynucleotidesequences is determined by direct comparison of the sequence informationbetween the molecules by aligning the sequences and determining theidentity by methods known in the art. In some embodiments, the percentidentity (e.g., amino acid sequence, nucleic acid sequence, and/or genesequence) is determined by a direct comparison of the sequenceinformation between two molecules by aligning the sequences, countingthe exact number of matches between the two aligned sequences, dividingby the length of the shorter sequence, and multiplying the result by100. Readily available computer programs find use in these analysesincluding those described above. Programs for determining nucleotidesequence identity are available in the Wisconsin Sequence AnalysisPackage, Version 8 (Genetics Computer Group, Madison, Wis.) for example,the BESTFIT, FASTA and GAP programs, which also rely on the Smith andWaterman algorithm. These programs are readily utilized with the defaultparameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above.

An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol., 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. These initial neighborhood word hits actas starting points to find longer HSPs containing them. The word hitsare expanded in both directions along each of the two sequences beingcompared for as far as the cumulative alignment score can be increased.Extension of the word hits is stopped when: the cumulative alignmentscore falls off by the quantity X from a maximum achieved value; thecumulative score goes to zero or below; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See,Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M′5, N′-4, and a comparisonof both strands.

The BLAST algorithm then performs a statistical analysis of thesimilarity between two sequences (See e.g., Karlin and Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 [1993]). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a serine proteasenucleic acid of this invention if the smallest sum probability in acomparison of the test nucleic acid to a serine protease nucleic acid isless than about 0.1, more preferably less than about 0.01, and mostpreferably less than about 0.001. Where the test nucleic acid encodes aserine protease polypeptide, it is considered similar to a specifiedserine protease nucleic acid if the comparison results in a smallest sumprobability of less than about 0.5, and more preferably less than about0.2.

In some embodiments of the present invention, sequences were analyzed byBLAST and protein translation sequence tools. In some experiments, thepreferred version was BLAST (Basic BLAST version 2.0). The programchosen was “BlastX”, and the database chosen was “nr.” Standard/defaultparameter values were employed.

Several methods are known in the art that are suitable for generatingvariant polynucleotide sequences of the POx enzymes of the presentinvention, include but are not limited to site-saturation mutagenesis,scanning mutagenesis, insertional mutagenesis, deletion mutagenesis,random mutagenesis, site-directed mutagenesis, and directed-evolution,as well as various other recombinatorial approaches.

This invention further provides expression vectors comprising at least afragment of the polynucleotides set forth above and host cells ororganisms transformed with these expression vectors. Useful vectorsinclude plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Accordingly, the invention alsoprovides a vector including a polynucleotide of the invention and a hostcell containing the polynucleotide. In general, the vector contains anorigin of replication functional in at least one organism, convenientrestriction endonuclease sites, and a selectable marker for the hostcell.

Preferably, a polynucleotide in a vector is operably linked to a controlsequence that is capable of providing for the expression of the codingsequence by the host cell, i.e. the vector is an expression vector. Thecontrol sequences may be modified, for example, by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators. The control sequences may in particularcomprise promoters.

In some embodiments, in the vector, the nucleic acid sequence encodingfor the POx signal peptide or the POx signal peptide fusion polypeptideis operably combined with a suitable promoter sequence. The promoter canbe any DNA sequence having transcription activity in the host organismof choice and can be derived from genes that are homologous orheterologous to the host organism. Examples of suitable promoters fordirecting the transcription of the modified nucleotide sequence, such asmodified enzyme nucleic acids, in a bacterial host include the promoterof the Streptomyces coelicolor agarase gene dagA promoters, GI and A4promoters, the promoters of the Bacillus licheniformis alpha-amylasegene (amyL), the aprE promoter of Bacillus subtilis, the promoters ofthe Bacillus stearothermophilus maltogenic amylase gene (amyM, thepromoters of the Bacillus amyloliquefaciens alpha-amylase gene (amyQ),the promoters of the Bacillus subtilis xyIA and xyIB genes and apromoter derived from a Lactococcus sp.-derived promoter including theP170 promoter.

A mature POx or a POx fusion polypeptide of the invention can beexpressed by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, e.g., byemploying blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thePOx polynucleotides can be synthesized by conventional techniquesincluding automated DNA synthesizers. Alternatively, PCR amplificationof gene fragments can be carried out using anchor primers that give riseto complementary overhangs between two consecutive gene fragments thatcan subsequently be annealed and reamplified to generate a chimeric genesequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, 1992). A POx signalpeptide-encoding nucleic acid can be cloned into an expression vectorsuch that the fusion moiety e.g. polypeptide of interest, is linkedin-frame to the POx signal peptide. An expression vector comprising apolynucleotide encoding a POx signal peptide can be any vector capableof expressing the polynucleotide encoding the mature form of the POxpolypeptide or the POx signal peptide fused to the mature portion of thePOx polypeptide in a selected host organism, and the choice of vectorwill depend on the host cell into which the expression vector isintroduced. Thus, in some embodiments, the invention provides expressionvectors that comprise a nucleotide sequence encoding a POx signalpeptide, as recited herein, operably linked to a nucleotide sequenceencoding a heterologous POx polypeptide. In another embodiment, thevectors of the invention comprise a polynucleotide sequence that encodesa POx polypeptide that lacks a signal peptide.

In some preferred embodiments, the POx polynucleotide is ligated into anappropriate expression plasmid. The cloned POx gene is then used totransform or transfect a host cell in order to express the POx gene.This plasmid may replicate in hosts in the sense that it contains thewell-known elements necessary for plasmid replication or the plasmid maybe designed to integrate into the host chromosome. The necessaryelements are provided for efficient gene expression (e.g., a promoteroperably linked to the gene of interest). In some embodiments, thesenecessary elements are supplied as the gene's own homologous promoter ifit is recognized, (i.e., transcribed, by the host), a transcriptionterminator (a polyadenylation region for eukaryotic host cells) which isexogenous or is supplied by the endogenous terminator region of the POxgene. In some embodiments, a selection gene such as an antibioticresistance gene that enables continuous cultural maintenance ofplasmid-infected host cells by growth in antimicrobial-containing mediais also included.

The mature POx polypeptides or POx fusion polypeptides may, in addition,can comprise a tag sequence that is fused to the C-terminus of the POxfusion polypeptide to generate a tagged POx fusion polypeptide. Such tagsequences can be used to identify transformants and/or to facilitate thepurification of recombinant Tat fusion polypeptides. For example, thePOx fusion polypeptide it may be expressed to contain a tag such asthose of maltose binding protein (MBP), glutathione-S-transferase (GST)or thioredoxin (TRX), or as a His tag. Kits for expression andpurification of such fusion proteins are commercially available from NewEngland BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) andInvitrogen, respectively. The POx fusion polypeptide can also be taggedwith an epitope and subsequently purified by using a specific antibodydirected to such epitope. One such epitope (“FLAG®) is commerciallyavailable from Kodak (New Haven, Conn.). Another tag that can be used inthe invention is the c-myc tag, as is described in the examples.

It is intended that although preferred enzymes for use in the vectorsand methods of the invention are referred to herein by their specificSEQ ID NOS, the present invention encompasses enzymes which are derivedfrom the nucleic acids which encode the corresponding amino acid SEQ IDNOS, when expressed, either in their native host species or aheterologous host species. Thus, the present invention encompassesembodiments in which the enzymes are co- or post-translationallyprocessed.

The chimeric polynucleotides of the invention encode POx fusion proteinsthat are directed to a secretory pathway which leads to the secretion ofthe mature forms of the POx enzymes. In some embodiments, the fusionpolypeptide comprises a Tat signal peptide that is the secretory leadersequence of polypeptides that are naturally expressed by Streptomycesand/or Bacillus that is operably linked to a mature form of a POxpolypeptide. In some embodiments, the signal peptide is a TAT signalpeptide. In other embodiments, the signal peptide is a SEC signalpeptide. In some embodiments, the fusion proteins of the inventioncomprise a signal peptide encoded by the S. coelicolor SCO6772 gene (SEQID NO:15), the signal sequence encoded by the S. coelicolor celA gene(EMBL accession no. AL939132.1; SEQ ID NO: 27); the signal sequenceencoded by the S. coelicolor secreted endoglucanase gene SCO7363 (SEQ IDNO:31), or the signal sequence encoded by the S. coelicolor possiblesecreted protein gene SCO0624 (SEQ ID NO: 39). In yet other embodiments,the fusion proteins of the invention comprise SEC signal peptide of theBacillus circulans cyclomaltodextrin glucanotransferase (cgt) precursor(Accession no. P43379; SEQ ID NO:36). It is understood that any signalpeptide capable of directing the fusion protein of the invention to asecretory pathway finds use in the present invention.

The mature forms of the POx enzymes of the invention can be the POxenzymes identified from a number of microorganisms including but notlimited to Streptomyces sp., Bacillus sp., Acidothermus sp., Arthobactersp., and marine Acinobacteium sp. In some preferred embodiments, theStreptomyces sp. include Streptomyces strain H-7775, Streptomyceslividans, Streptomyces avermitilis, Streptomyces coelicolor andStreptomyces species IKD472. In other preferred embodiments, theAcidothermus sp. is Acidothermus cellulolyticus 11B (Q2E2H5). In someembodiments, the Arthobacter sp. is Arthrobacter sp. FB24 (Q4NJLO). Insome embodiments, the marine Actinobacterium is ActinobacteriumPHSC20C1.

POx enzymes encompassed by the invention include homologous orheterologous POx proteins. POx enzymes of interest include full-lengthPOx polypeptides that are naturally synthesized with a signal peptide,the mature form of the full-length POx polypeptides, and POxpolypeptides that naturally lack a signal peptide.

The mature POx polypeptides and/or the POx fusion polypeptides may, inaddition, comprise a tag sequence that is fused to the C-terminus of themature POx polypeptide and the POx fusion polypeptide to generate atagged mature POx polypeptide of POx fusion polypeptide. Such tagsequences can be used to identify transformants and/or to facilitate thepurification of recombinant POx fusion polypeptides.

For example, the POx fusion polypeptide it may be expressed to contain atag such as those of maltose binding protein (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag.Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The Tatfusion polypeptide can also be tagged with an epitope and subsequentlypurified by using a specific antibody directed to such epitope. One suchepitope (“FLAG®) is commercially available from Kodak (New Haven,Conn.). Another tag that can be used in the invention is the c-myc tag.

Some preferred embodiments of fusion proteins include the fusion proteinof SEQ ID NO:16 which comprises the mature form of Streptomyces speciesH-7775 (SEQ ID NO:2) operably linked (fused) to the signal peptide ofStreptomyces coelicolor gene SCO6772 (SEQ ID NO:15); the fusion proteinof SEQ ID NO:28 which comprises the mature form of Streptomyces speciesH-7775 (SEQ ID NO:2) operably linked (fused) to the signal peptide ofStreptomyces coelicolor celA gene (EMBL Accession No. AL939132.1; SEQ IDNO:27). Other preferred embodiments of fusion proteins include thefusion protein of SEQ ID NO:38, which comprises the signal peptide ofthe Bacillus cgt precursor (SEQ ID NO:36) fused to the mature form ofthe POx protein of Streptomyces species H-7775 (SEQ ID NO:2); the fusionprotein of SEQ ID NO:35, which comprises the signal peptide of thesecreted endoglucanase from Streptomyces coelicolor SCO 7363 (SEQ IDNO:31) fused to the mature form of the POx protein of Acidothermuscellulolyticus (SEQ ID NO:6); the fusion protein of SEQ ID NO:37, whichcomprises the signal peptide of the signal peptide of the Bacillus cgtprecursor (SEQ ID NO:36) fused to the mature form of the POx protein ofAcidothermus cellulolyticus (SEQ ID NO:6); the fusion protein of SEQ IDNO:40, which comprises the signal peptide of the secreted proteinStreptomyces coelicolor SCO0624 (SEQ ID NO:39) fused to the mature formof the POx protein of Arthrobacter sp. gene FB24 (Q4NJLO; SEQ ID NO:11).

In some embodiments, the POx enzyme of the invention is expressedwithout a signal peptide. Any POx expressed in the absence of a signalpeptide will not be translocated to the outside of the host cell, butwill remain confined to the intracellular milieu. Examples of POxenzymes that are expressed intracellularly include the POx enzyme fromStreptomyces sp. H-7775 (SEQ ID NO:2), the POx enzyme of Streptomyceslividans (SEQ ID NO:4) and the POx enzyme of Acidothermus sp. 11 B (SEQID NO:6).

The invention encompasses POx proteins that are related to the POxpolypeptide of SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,16, 28,33, 35, 37, 38, and/or 40. In some embodiments, the inventionencompasses variant proteins of SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14,16, 28, 33, 35, 37, 38, and/or 40. In some preferredembodiments, variant proteins differ from a parent protein and oneanother by a small number of amino acid residues. The number ofdiffering amino acid residues may be one or more, preferably 1, 2, 3, 4,5, 10, 15, 20, 30, 40, 50, or more amino acid residues. In somepreferred embodiments, the number of different amino acids betweenvariants is between 1 and 10. In some particularly preferredembodiments, related proteins and particularly variant proteins share atleast about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,about 97%, about 98%, or about 99% amino acid sequence identity with theamino acid sequence of the precursor POx and have comparable or enhancedPOx activity of the precursor polypeptide.

In preferred embodiments, the invention encompasses variants of POxproteins of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,16, 28,33, 35, 37, 38, and/or 40. As used herein, the term “variant(s)” as usedin context of a polypeptide sequence refers to a polypeptide preparedfrom the original (i.e., parent) polypeptide, or by using the sequenceinformation from the original (i.e., parent) polypeptide, by insertion,deletion and/or substitution of one or more amino acids in the original(i.e., parent) sequence. In some embodiments, at least one insertion,deletion, and/or substitution is made in the original (i.e., parent)sequence, while in other embodiments, preferably less than about 50amino acids, less than about 40, less than about 30, less than about 20,or less than about 10 amino acids are modified by insertion, deletionand/or substitution of the original (i.e., parent) sequence in order toproduce variant polypeptides. In some preferred embodiments, only oneamino acid modification (i.e., insertion, deletion or substitution) ismade, while in other preferred embodiments two amino acids are modified,and in still further embodiments, three amino acids are modified, and inyet additional embodiments, four amino acids are modified, and in stillfurther embodiments, five amino acids are modified. It is not intendedthat the variants of the present invention be limited to any specificnumber nor type of amino acid modifications.

In other preferred embodiments, the invention encompasses homologues ofthe POx polypeptides of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14,16, 28, 33, 35, 37, 38, and/or 40. The homologs of the POxpolypeptides have are at least about 70% homologous, more preferably atleast about 80% homologous, still more preferably at least about 85%homologous, further more preferably at least about 90% homologous, evenmore preferably at least about 95%, more preferably about 96%, stillmore preferably about 97%, even more preferably about 98%, or mostpreferably about 99% homologous to the polypeptide sequence of interest(e.g., a sorbitol oxidase provided by the present invention). In someembodiments, homology between two polypeptide sequences is determinedusing ClustalW alignment algorithm using standard settings, as referredto herein. However, it is not intended that the present invention belimited to any particular method for determining homology.

The variants, homologues and fragments provided by the present inventionall retain at least some of the desired enzymatic activity of the parentenzyme, such as at least about 10%, at least about 20%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80% at least about 90%, or all theenzyme activity of the parent enzyme. In some alternative embodiments,the variants and/or homologues have greater enzymatic activity than theoriginal (i.e., parent) enzyme.

The POx enzymes of the present invention were found to be thermallystable and stable over a wide pH range. For example the POx of SEQ IDNO:4 was found to have SOx activity at pH ranging from pH3.5 to 5.5,from pH 5.5-6.7, from pH 6-8, and from pH 7.6-9.0 at a temperature of25° C. In preferred embodiments, the POx enzyme of the inventionexhibits maximal activity at pH 5.5, pH 6.0 and pH 8.0. The POx of theinvention retain at least 60% activity, more preferable at least 70%activity, more preferably at least 80% activity, more preferably atleast 90% activity, more preferably at least 95% activity. Indeed, thepH profiles of the sorbitol oxidases of the present invention arecompatible with the pHs necessarily used in industry, as well asdetergents and other cleaning agents.

It is contemplated that the oxidases of the present invention will finduse in numerous applications, including but not limited to cleaningcompositions (e.g., laundry and dish detergents, etc.), personal care(e.g., oral care, skin care, etc.), textile processing, diagnostics(e.g., medical diagnostic methods), biosensors, and other suitableapplications.

Suitable host cells for use in the present invention are members ofthose genera capable of being utilized for industrial biosyntheticproduction of desired POx enzymes. Accordingly, host cells can includeprokaryotes belonging to the genera Escherichia, Corynebacterium,Brevibacterium, Acidothermus, Arthrobacter, Bacillus, Pseudomonas,Streptomyces, Staphylococcus, or Serratia. Eukaryotic host cells canalso be utilized, with yeasts of the genus Saccharomyces orSchizosaccharomyces being included.

More specifically, prokaryotic host cells suitable for use in thepresent invention include, but are not limited to, Escherichia coli,Bacillus brevis, Bacillus cereus, Bacillus mesentericus, B.licheniformis, B. lentus, B. subtilis, B. amyloliquefaciens, B. lentus,B. brevis, B. stearothermophilus, B. alkalophilus, B. coagulans, B.circulans, B. pumilus, B. thuringiensis, B. clausii, B. megaterium, S.lividans, Streptomyces aureofaciens, Strepbomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, Streptomyces kasugensis,Streptomyces murinus, S. rubiginosus, and S. griseus. In preferredembodiments, the host cells for use in the present invention includeStreptomyces sp. H-7775, S. lividans, E. coli, Acidothermus and B.subtilis. In some preferred embodiments, the host cells areStreptomyces, in other embodiments, the host cells are E. coli, in yetother embodiments, the host cells are Acidothermus, and in still otherembodiments, the host cells are Bacillus. Indeed, it is not intendedthat the present invention be limited to any particular species of hostcells, as various organisms find use as host cells of the presentinvention.

The present invention provides host cells comprising a recombinantexpression vector comprising at least one polynucleotide sequenceencoding at least one polypeptide selected from SEQ ID NOS:2, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35, 37, 38, and/or 40 orhomologues, fragments or variants thereof. In alternative embodiments,the host cells comprise at least one polynucleotide sequence encoding atleast one polypeptide selected from SEQ ID NOS: SEQ ID NOS:2, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35, 37, 38, and/or 40 orhomologues, fragments or variants thereof. In some embodiments,polynucleotide sequence encoding at least one polypeptide selected fromSEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35,37, 38, and/or 40 or homologues, fragments or variants thereof ispresent in the genome of the host cell. In other embodiments,polynucleotide sequence encoding at least one polypeptide selected fromSEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35,37, 38, and/or 40 or homologues, fragments or variants thereof ispresent in a vector that replicates autonomously in the host cell.

In some embodiments, the invention provides E. coli host cellscomprising a polynucleotide that encodes a POx polypeptide of SEQ IDNOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35, 37, 38,and/or 40. In preferred embodiments, the E. coli host cell comprises aPOx polypeptide that encodes a POx protein of SEQ ID NO:2 or 4. In otherembodiments, host cells of the invention are S. lividans cells thatcomprise a polynucleotide that encodes a POx polypeptide of SEQ IDNOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35, 37, 38,and/or 40. In preferred embodiments, the S. lividans host cell comprisesa POx polypeptide that encodes a POx protein of SEQ ID NO:2, 4, 6 and/or11, 16, 28, 35 and/or 40. In yet other embodiments, host cells of theinvention are B. subtilis cells that comprise a polynucleotide thatencodes a POx polypeptide of SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 28, 33, 35, 37, 38, and/or 40. In preferred embodiments,the B. subtilis host cell comprises a POx polypeptide that encodes a POxprotein of SEQ ID NO:2, 6 and/or 38.

In some preferred embodiments, the expression of the at least onepolypeptide having oxidase activity is extracellular, while in otherembodiments, the expression is intracellular.

Host cells transformed with POx polynucleotide sequences encodingheterologous or homologous protein may be cultured under conditionssuitable for the expression and recovery of the encoded protein fromcell culture.

The present invention provides methods of using the above-describedcells for the production of at least one recombinant POx polypeptide. Insome embodiments, the methods include culturing a host cell of theinvention to produce a POx protein. In some embodiments, the methodsinclude providing a recombinant expression vector that contains a POxpolynucleotide that encodes a POx enzyme; transforming the host cellwith the expression vector to produce a transformed host cell; andgrowing the transformed host cell under conditions suitable for theexpression of the at least one polypeptide; and recovering the at leastone polypeptide expressed by the transformed host cell. In someadditional embodiments and as discussed above, the protein is secretedinto the culture medium. In yet further embodiments, the methodscomprise the step of recovering the protein from the culture medium.Alternatively, the methods comprise the step of recovering the proteinfrom within the host cell. Preferred embodiments provide for theproduction of at least one recombinant polypeptide, wherein said atleast one polypeptide exhibits oxidase activity, wherein the polypeptidecomprises SEQ ID NOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,28, 33, 35, 37, 38, and/or 40 or homologues, fragments or variantsthereof, comprising: (a) providing: a recombinant expression vector,wherein said expression vector comprises at least one polynucleotidesequence encoding at least one of said polypeptides selected from SEQ IDNOS:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35, 37, 38,and/or 40 or homologues, fragments or variants thereof; and a host cell;(b) transforming the host cell with the expression vector to produce atransformed host cell; (c) growing the transformed host cell underconditions suitable for the expression of at least one polypeptide; and(d) recovering at least one polypeptide expressed by the transformedhost cell. In some preferred embodiments, the oxidase activity is polyoloxidase activity. In some particularly preferred embodiments, theoxidase activity is sorbitol oxidase activity, while in some alternativeparticularly preferred embodiments, the oxidase activity is xylitoloxidase activity.

In some preferred embodiments, the POx protein expressed by the hostcells is recovered by the removal of other host cell constituents in thegrowth media using any convenient method (e.g., by precipitation,centrifugation, affinity, filtration) or any other suitable method knownin the art. For example, affinity chromatography (Tilbeurgh et al., FEBSLett., 16:215 [1984]); ion-exchange chromatographic methods (Goya) etal., Biores. Technol., 36:37 [1991]; Fliess et al., Eur. J. Appl.Microbiol. Biotechnol. 17:314 [1983]; Bhikhabhai et al., J. Appl.Biochem., 6:336 [1984]; and Ellouz et al., Chromatography 396:307[1987]), including ion-exchange using materials with high resolutionpower (Medve et al., J. Chromatography A 808:153 [1998]; hydrophobicinteraction chromatography (Tomaz and Queiroz, J. Chromatography A865:123 [1999]; two-phase partitioning (Brumbauer et al., Bioseparation7:287 [1999]); ethanol precipitation; reverse phase HPLC; chromatographyon silica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; and gel filtration (e.g.,using SEPHADEX G-75), find use in the present invention. In particularlypreferred embodiments, the sorbitol oxidases of the present inventionare substantially purified to a level of at least about 99% of theprotein component, as determined by SDS-PAGE or other standard methodsknown in the art.

The invention also encompasses POx fusion polypeptides that comprise asignal peptide and a heterologous peptide and a polypeptide domain thatwill facilitate purification of POx enzymes (Kroll D J et al (1993) DNACell Biol 12:441-53). Such purification facilitating domains include,but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals (Porath J (1992) Protein Expr Purif 3:263-281), protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequence suchas Factor XA or enterokinase (Invitrogen, San Diego Calif.) between thepurification domain and the heterologous protein can be used tofacilitate purification. In some particular embodiments, the sorbitoloxidase is used without purification from the other components in theculture medium. In some embodiments, the components of the culturemedium are simply concentrated and then used without furtherpurification of the POx protein from the other components of the growthmedium in order to produce a cleaning and/or other composition.

In some embodiments, recombinant POx are expressed in bacterial orfungal host cells and these recombinant POx are purified by the removalof other host cell constituents; the percent of recombinant POxpolypeptides is thereby increased in the sample. In particularlypreferred embodiments, the POx of the present invention aresubstantially purified to a level of at least about 99% of the proteincomponent, as determined by SDS-PAGE or other standard methods known inthe art.

Means for determining the levels of secretion of a heterologous orhomologous protein in a gram-positive host cell and detecting secretedproteins include, using either polyclonal or monoclonal antibodiesspecific for the protein. Examples include enzyme-linked immunosorbentassay (ELISA), radioimmunoassay (RIA) and fluorescent activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton R et al (1990, Serological Methods, a LaboratoryManual, APS Press, St Paul Minn.) and Maddox D E et al (1983, J Exp Med158:1211).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and can be used in various nucleic and amino acidassays. Means for producing labeled hybridization or PCR probes fordetecting specific polynucleotide sequences include oligo labeling, nicktranslation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the nucleotide sequence, or any portion ofit, may be cloned into a vector for the production of an mRNA probe.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides.

A number of companies such as Pharmacia Biotech (Piscataway N.J.),Promega (Madison Wis.), and US Biochemical Corp (Cleveland Ohio) supplycommercial kits and protocols for these procedures. Suitable reportermolecules or labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles and the like. Patents teachingthe use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. The enzymaticactivity of a secreted SOx-fusion polypeptide can be determined bycontacting the secreted polypeptide with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate.In addition, verifying that the correct enzyme or other reportermolecule is secreted can be accomplished by performing mass spectroscopyof the secreted protein.

In some embodiments, the host cells are cultured under batch, fed-batchor continuous fermentation conditions. Classical batch fermentationmethods use a closed system, wherein the culture medium is made prior tothe beginning of the fermentation run, the medium is inoculated with thedesired organism(s), and fermentation occurs without the subsequentaddition of any components to the medium. In certain cases, the pH andoxygen content, but not the carbon source content, of the growth mediumis altered during batch methods. The metabolites and cell biomass of thebatch system change constantly up to the time the fermentation isstopped. In a batch system, cells usually progress through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase eventually die. In general terms, the cells in logphase produce most protein.

A variation on the standard batch system is the “fed-batch fermentation”system. In this system, nutrients (e.g., a carbon source, nitrogensource, O₂, or other nutrient) are only added when their concentrationin culture falls below a threshold. Fed-batch systems are useful whencatabolite repression is apt to inhibit the metabolism of the cells andwhere it is desirable to have limited amounts of nutrients in themedium. Measurement of the actual nutrient concentration in fed-batchsystems is estimated on the basis of the changes of measurable factorssuch as pH, dissolved oxygen and the partial pressure of waste gasessuch as CO₂. Batch and fed-batch fermentations are common and known inthe art.

Continuous fermentation is an open system where a defined culture mediumis added continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth and/or end productconcentration. For example, in some embodiments, a limiting nutrientsuch as the carbon source or nitrogen source is maintained at a fixedrate and all other parameters are allowed to moderate. In other systems,a number of factors affecting growth are altered continuously while thecell concentration, measured by media turbidity, is kept constant.Continuous systems strive to maintain steady state growth conditions.Thus, cell loss due to medium being drawn off may be balanced againstthe cell growth rate in the fermentation. Methods of modulatingnutrients and growth factors for continuous fermentation processes aswell as techniques for maximizing the rate of product formation areknown to those of skill in the art and find use in the production of thePOx enzymes of the present invention.

The POx protein produced using the above described methods finds use inany product containing a POx, including, but not limited to cleaningcompositions, (e.g., fabric cleaning compositions, such as laundrydetergents, surface cleaning compositions, dish cleaning compositionsand automatic dishwasher detergent compositions; See e.g., WO0001826,which is incorporated by reference herein), bleaching and disinfectingcompositions.

The cleaning compositions and cleaning additives of the presentinvention require an effective amount of the POx enzymes provided by thepresent invention. The required level of enzyme may be achieved by theaddition of one or more POx enzymes, variants, homologues, and/or otherenzymes or enzyme fragments having the activity of the POx enzymes ofthe present invention. Typically, the cleaning compositions of thepresent invention comprise at least 0.1 ppm to about 10 ppm, from about0.5 to about 2 ppm, or from about 1 to about 5 ppm. In some preferredembodiments, the cleaning compositions of the present invention compriseabout 0.2 to about 1 ppm of POx enzymes. The cleaning compositions thatcomprise at least one POx enzyme in combination with a perhydrolaseenzyme comprise an effective amount of POx enzyme as recited herein andan effective amount of perhydrolase that comprises about 0.1 to about 10ppm, about 0.5 to about 10 ppm or about 1 to about 5 ppm ofperhydrolase. In preferred embodiments, the amount of perhydrolasecontained in the cleaning compositions is from about 0.2 or about 1 ppm.In some embodiments, the cleaning compositions comprise a perhydrolasesubstrate as recited above in amounts from about 0.2 to about 20 mM, orfrom about 0.5 to about 15 mM, from about 1 to about 10 mM. In preferredembodiments, the amount of perhydrolase substrate contained in thecleaning composition comprises from about 1 to about 5 mM ofperhydrolase substrate.

In some particular embodiments, the POx protein is used in anPOx-containing laundry detergent comprising from about 1% to about 80%,e.g., about 5% to about 50% (by weight) of surfactant, which may be anon-ionic surfactant, cationic surfactant, an anionic surfactant or azwitterionic surfactant, or any mixture thereof (e.g., a mixture ofanionic and nonionic surfactants). Exemplary surfactants include: alkylbenzene sulfonate (ABS), including linear alkyl benzene sulfonate andlinear alkyl sodium sulfonate, alkyl phenoxy polyethoxy ethanol (e.g.,nonyl phenoxy ethoxylate or nonyl phenol), diethanolamine,triethanolamine and monoethanolamine. Exemplary surfactants that finduse in laundry detergents are known in the art (See e.g., U.S. Pat. Nos.3,664,961, 3,919,678, 4,222,905, and 4,239,659).

The laundry detergent may be in solid, liquid, gel or bar form, and mayfurther contain a buffer such as sodium carbonate, sodium bicarbonate,or detergent builder, bleach, bleach activator, various enzymes, anenzyme stabilizing agent, suds booster, suppresser, anti-tarnish agent,anti-corrosion agent, soil suspending agent, soil release agent,germicide, pH adjusting agent, non-builder alkalinity source, chelatingagent, organic or inorganic filler, solvent, hydrotrope, opticalbrightener, dye or perfumes. In some preferred embodiments, the laundrydetergent comprises in addition to the POx enzymes of the presentinvention, at least one further enzyme (e.g., hemicellulase, peroxidase,protease, cellulase, xylanase, lipase, phospholipase, esterase,cutinase, pectinase, keratinase, reductase, oxidase, phenoloxidase,lipoxygenase, ligninase, pullulanase, tannase, pentosanase, mannanase,B-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, andamylases, or mixtures thereof).

The POx protein of the present invention finds use in any suitablecomposition useful for cleaning a variety of surfaces in need of stainremoval. Such cleaning compositions include detergent compositions forcleaning hard surfaces, unlimited in form (e.g., liquid, gel, bar andgranular formation); detergent compositions for cleaning fabrics,unlimited in form (e.g., granular, liquid, gel and bar formulations);dishwashing compositions (unlimited in form); oral cleaningcompositions, unlimited in form (e.g., dentifrice, toothpaste, gel andmouthwash formulations); denture cleaning compositions, unlimited inform (e.g., liquid, gel or tablet); and contact lens cleaningcompositions, unlimited in form (e.g., liquid, tablet).

In some embodiments, the cleaning compositions of the present inventioncomprise an effective amount of a POx, alone or in combination withother POx enzymes and/or a perhydrolase enzyme as recited above. In oneembodiment, the cleaning composition comprises a POx of SEQ ID NO:2, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35, 37, 38, and/or40. In one embodiment, the cleaning composition comprises a POx of SEQID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16. In onepreferred embodiment, the cleaning composition comprises a POx of SEQ IDNO:2, 4, 6, and/or 11.

In yet other embodiments, the cleaning composition comprises a POx ofSEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 33, 35,37, 38, and/or 40 in combination with at least one additional enzymeincluding enzymes that are hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, keratinases, reductases, oxidases, oxidoreductases,perhydrolases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, mannanases, β-glucanases, arabinosidases,hyaluronidases, chondroitinases, laccases, and amylases. In yet otherembodiments, the cleaning composition comprises a POx of SEQ ID NO:2, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 in combination with atleast one additional enzyme including enzymes that are hemicellulases,peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, keratinases, reductases, oxidases,oxidoreductases, perhydrolases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, mannanases,β-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases,and amylases. In another preferred embodiments, the cleaning compositioncomprises a POx of SEQ ID NO:2, 4, 6, and/or 11 in combination with atleast one additional enzyme including enzymes that are hemicellulases,peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, keratinases, reductases, oxidases,oxidoreductases, perhydrolases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, mannanases,β-glucanases, arabinosidases, hyaluronidases, chondroitinases, laccases,and amylases. In another embodiment, the cleaning composition comprisesa POx of SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28,33, 35, 37, 38, and/or 40 in combination with a perhydrolase. In anotherembodiment, the cleaning composition comprises a POx of SEQ ID NO:2, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 in combination with aperhydrolase. In a preferred embodiment, the cleaning compositioncomprises a POx of SEQ ID NO:2, 4, 6, and/or 11 in combination with aperhydrolase. In another embodiment, the cleaning composition comprisesa POx of SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28,33, 35, 37, 38, and/or 40 in combination with a bleach activator. Inanother embodiment, the cleaning composition comprises a POx of SEQ IDNO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and/or 16 in combinationwith a bleach activator. In a preferred embodiment, the cleaningcomposition comprises a POx of SEQ ID NO:2, 4, 6, and/or 11 incombination with a bleach activator.

In some embodiments, the cleaning compositions of the present inventioncomprise one or more detergent enzymes which provide cleaning, bleachingand/or disinfecting benefits. Examples of suitable enzymes include, butare not limited to, hem icellulases, peroxidases, proteases, cellulases,xylanases, lipases, phospholipases, esterases, cutinases, pectinases,keratinases, reductases, carrageenases, phenoloxidases, lipoxygenases,ligninases, pullulanases, tannases, pentosanases, mannanases,β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase,and amylases, or mixtures thereof. In some embodiments, a combination ofenzymes is used (i.e., a “cocktail”) comprising conventional applicableenzymes like protease, lipase, cutinase and/or cellulase in conjunctionwith the POx or POx-perhydrolase combination in the presence or absenceof a bleach activator.

In some embodiments, the cleaning compositions also comprise, inaddition to the proteins described herein, one or more cleaningcomposition materials compatible with the POx protein. As describedherein, the term “cleaning composition material,” refers any liquid,solid or gaseous material selected for the particular type of cleaningcomposition desired and the form of the product (e.g., liquid, granule,bar, spray, stick, paste, gel), which materials are also compatible withthe POx or POx-perhydrolase combinations used in the composition eitherin the presence or absence of a bleach activator. The specific selectionof cleaning composition materials are readily made by considering thesurface material to be cleaned, the desired form of the composition forthe cleaning condition during use (e.g., through the wash detergentuse). As used herein, “non-fabric cleaning compositions” include hardsurface cleaning compositions, dishwashing compositions, oral cleaningcompositions, denture cleaning compositions and contact lens cleaningcompositions.

The POx protein finds use with various conventional ingredients toprovide fully-formulated hard-surface cleaners, dishwashingcompositions, fabric laundering compositions, and the like. Suchcompositions find use in the form of liquids, granules, bars and thelike. In some embodiments, such compositions are formulated as modern“concentrated” detergents which contain as much as about 30% to about60% by weight of surfactants.

In some embodiments, the cleaning compositions of the present inventioncomprise various anionic, nonionic, zwitterionic, etc., surfactants.Such surfactants are typically present at levels of from about 5% toabout 35% of the compositions. Surfactants include nonionic, anionic,cationic, anionic or zwitterionic detergents (See e.g., U.S. Pat. Nos.4,404,128 and 4,261,868). A suitable detergent formulation is thatdescribed in U.S. Pat. No. 5,204,015 (previously incorporated byreference). Those in the art are familiar with the differentformulations which find use as cleaning compositions. As indicatedabove, in some preferred embodiments, the detergent compositions of thepresent invention employ a surface active agent (i.e., surfactant)including anionic, non-ionic and ampholytic surfactants well known fortheir use in detergent compositions. Some surfactants suitable for usein the present invention are described in British Patent Application No.2 094 826 A, incorporated herein by reference. In some embodiments,mixtures surfactants are used in the present invention.

Suitable anionic surfactants for use in the detergent composition of thepresent invention include linear or branched alkylbenzene sulfonates;alkyl or alkenyl ether sulfates having linear or branched alkyl groupsor alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates; alkanesulfonates and the like. Suitable counter ions for anionic surfactantsinclude alkali metal ions such as sodium and potassium; alkaline earthmetal ions such as calcium and magnesium; ammonium ion; andalkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.

Ampholytic surfactants that find use in the present invention includequaternary ammonium salt sulfonates, betaine-type ampholyticsurfactants, and the like. Such ampholytic surfactants have both thepositive and negative charged groups in the same molecule.

Nonionic surfactants that find use in the present invention generallycomprise polyoxyalkylene ethers, as well as higher fatty acidalkanolamides or alkylene oxide adduct thereof, fatty acid glycerinemonoesters, and the like.

In some preferred embodiments, the surfactant or surfactant mixtureincluded in the detergent compositions of the present invention isprovided in an amount from about 1 weight percent to about 95 weightpercent of the total detergent composition and preferably from about 5weight percent to about 45 weight percent of the total detergentcomposition. In various embodiments, numerous other components areincluded in the compositions of the present invention. Many of these aredescribed below. It is not intended that the present invention belimited to these specific examples. Indeed, it is contemplated thatadditional compounds will find use in the present invention. Thedescriptions below merely illustrate some optional components.

In some embodiments of the present invention, the composition containsfrom about 0 to about 50 weight percent of one or more buildercomponents selected from the group consisting of alkali metal salts andalkanolamine salts of the following compounds: phosphates, phosphonates,phosphonocarboxylates, salts of amino acids, aminopolyacetates highmolecular electrolytes, non-dissociating polymers, salts of dicarboxylicacids, and aluminosilicate salts. Examples of suitable divalentsequestering agents are disclosed in British Patent Application No. 2094 826 A, the disclosure of which is incorporated herein by reference.

In additional embodiments, compositions of the present invention containfrom about 1 to about 50 weight percent, preferably from about 5 toabout 30 weight percent, based on the composition of one or more alkalimetal salts of the following compounds as the alkalis or inorganicelectrolytes: silicates, carbonates and sulfates as well as organicalkalis such as triethanolamine, diethanolamine, monoethanolamine andtriisopropanolamine.

The cleaning compositions herein may contain a chelating agent, Suitablechelating agents include copper, iron and/or manganese chelating agentsand mixtures thereof. When a chelating agent is used, the cleaningcomposition may comprise from about 0.1% to about 15% or even from about3.0% to about 10% chelating agent by weight of the subject cleaningcomposition.

In yet additional embodiments of the present invention, the compositionscontain from about 0.1 to about 5 weight percent of one or more of thefollowing compounds as antiredeposition agents: polyethylene glycol,polyvinyl alcohol, polyvinylpyrrolidone and carboxymethylcellulose. Insome preferred embodiments, a combination of carboxymethyl-celluloseand/or polyethylene glycol are utilized with the composition of thepresent invention as useful dirt removing compositions.

The use of the POx enzymes of the present invention are used incombination with additional bleaching agent(s) such as sodiumpercarbonate, sodium perborate, sodium sulfate/hydrogen peroxide adductand sodium chloride/hydrogen peroxide adduct and/or a photo-sensitivebleaching dye such as zinc or aluminum salt of sulfonated phthalocyaninefurther improves the detergent effects. In additional embodiments, thePOx enzymes of the present invention are used in combination with bleachboosters (e.g., TAED and/or NOBS).

The cleaning compositions can comprise one or more detergent enzymeswhich provide cleaning performance and/or fabric care benefits. Examplesof suitable enzymes include, but are not limited to, hemicellulases,peroxidases, proteases, cellulases, xylanases, lipases, phospholipases,esterases, cutinases, pectinases, keratinases, reductases, oxidases,phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases,pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase,chondroitinase, laccase, and amylases, or mixtures thereof.

In addition to the ingredients described above, perfumes, buffers,preservatives, dyes and the like also find use with the presentinvention. Other ingredients useful in detergent cleaning compositionsalso find use in the compositions herein, including other activeingredients, carriers, hydrotropes, processing aids, dyes or pigments,solvents for liquid formulations, etc. If an additional increment ofsudsing is desired, suds boosters such as the C₁₀-C₁₆ alkylamides alsofind use in the compositions, typically at about 1% to about 10% levels.

In some embodiments, the detergent compositions comprise water and/orother solvents as carriers. For example, in some embodiments, lowmolecular weight primary or secondary alcohols (e.g., methanol, ethanol,propanol, and isopropanol) are suitable. Monohydric alcohols arepreferred for solubilizing surfactants, but polyols such as thosecontaining from about 2 to about 6 carbon atoms and from about 2 toabout 6 hydroxy groups (e.g., 1,3-propanediol, ethylene glycol,glycerine, and 1,2-propanediol) also find use. In such embodiments, thecompositions typically contain from about 5% to about 90%, or typicallyfrom about 10% to about 50% of such carriers.

In some embodiments, the detergent compositions provided herein areformulated such that during use in aqueous cleaning operations, the washwater will have a pH between about 5.5 and about 10.5. In some preferredembodiments, the pH of the wash water is between about pH 5 and pH 7. Inother preferred embodiments the pH of the wash water is between about pH7 and pH 10.5. Finished products thus are typically formulated at thisrange. Techniques for controlling pH at recommended usage levels includethe use of buffers, alkalis, acids, etc., and are well known to thoseskilled in the art.

Various bleaching compounds, such as the percarbonates, perborates andthe like, also find use in such compositions, typically at levels fromabout 1% to about 15% by weight. In some embodiments, such compositionsalso contain bleach activators such as tetraacetyl ethylenediamine,nonanoyloxybenzene sulfonate, and the like, which are also known in theart. Usage levels typically range from about 1% to about 10% by weight.

Various soil release agents, especially of the anionic oligoester type,various chelating agents, especially the aminophosphonates andethylenediaminedisuccinates, various clay soil removal agents,especially ethoxylated tetraethylene pentamine, various dispersingagents, especially polyacrylates and polyasparatates, variousbrighteners, especially anionic brighteners, various suds suppressors,especially silicones and secondary alcohols, various fabric softeners,especially smectite clays, and the like, all find use in variousembodiments of the present compositions, at levels ranging from about 1%to about 35% by weight. Standard formularies and published patentscontain multiple, detailed descriptions of such conventional materials.

Enzyme stabilizers also find use in the cleaning compositions of thepresent invention. Such stabilizers include, but are not limited topropylene glycol (preferably from about 1% to about 10%), sodium formate(preferably from about 0.1% to about 1%), and calcium formate(preferably from about 0.1% to about 1%).

The cleaning compositions of the present invention are formulated intoany suitable form and prepared by any suitable process chosen by theformulator, (See e.g., U.S. Pat. No. 5,879,584, U.S. Pat. No. 5,691,297,U.S. Pat. No. 5,574,005, U.S. Pat. No. 5,569,645, U.S. Pat. No.5,565,422, U.S. Pat. No. 5,516,448, U.S. Pat. No. 5,489,392, U.S. Pat.No. 5,486,303, U.S. Pat. No. 4,515,705, U.S. Pat. No. 4,537,706, U.S.Pat. No. 4,515,707, U.S. Pat. No. 4,550,862, U.S. Pat. No. 4,561,998,U.S. Pat. No. 4,597,898, U.S. Pat. No. 4,968,451, U.S. Pat. No.5,565,145, U.S. Pat. No. 5,929,022, U.S. Pat. No. 6,294,514, and U.S.Pat. No. 6,376,445, all of which are incorporated herein by referencefor some non-limiting examples). When formulating the hard surfacecleaning compositions and fabric cleaning compositions of the presentinvention, the formulator may wish to employ various builders at levelsfrom about 5% to about 50% by weight. Typical builders include the 1-10micron zeolites, polycarboxylates such as citrate and oxydisuccinates,layered silicates, phosphates, and the like. Other conventional buildersare listed in standard formularies.

Other optional ingredients include chelating agents, clay soilremoval/anti-redeposition agents, polymeric dispersing agents, bleaches,brighteners, suds suppressors, solvents and aesthetic agents.

In some preferred embodiments, the cleaning compositions of the presentinvention find use in cleaning surfaces and/or fabrics. In someembodiments, at least a portion of the surface and/or fabric iscontacted with at least one embodiment of the cleaning compositions ofthe present invention, in neat form or diluted in a wash liquor, andthen the surface and/or fabric is optionally washed and/or rinsed. Forpurposes of the present invention, “washing” includes, but is notlimited to, scrubbing, and mechanical agitation. In some embodiments,the fabric comprises any fabric capable of being laundered in normalconsumer use conditions. In some preferred embodiments, the cleaningcompositions of the present invention are used at concentrations of fromabout 500 ppm to about 15,000 ppm in solution. In some embodiments inwhich the wash solvent is water, the water temperature typically rangesfrom about 5° C. to about 70° C. In some preferred embodiments forfabric cleaning, the water to fabric mass ratio is typically from about1:1 to about 30:1.

In order to further illustrate the present invention and advantagesthereof, the following specific Examples are given with theunderstanding that they are being offered to illustrate the presentinvention and should not be construed in any way as limiting its scope.

EXPERIMENTAL

The following Examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: ° C. (degrees Centigrade); rpm (revolutions perminute); H₂O (water); HCl (hydrochloric acid); aa (amino acid); by (basepair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg and ug(micrograms); mg (milligrams); ng (nanograms); μl and ul (microliters);ml (milliliters); mm (millimeters); nm (nanometers); μm and um(micrometer); M (molar); mM (millimolar); μM and uM (micromolar); U(units); V (volts); MW (molecular weight); sec (seconds); min(s)(minute/minutes); hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl(sodium chloride); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (opticaldensity at 600 nm); EFT (“effective fermentation time”); HDL (Heavy DutyDetergent Liquid); EtOH (ethanol); PBS (phosphate buffered saline [150mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); SDS (sodium dodecylsulfate); Tris (tris(hydroxymethyl)aminomethane); TAED(N,N,N′N′-tetraacetylethylenediamine); w/v (weight to volume); v/v(volume to volume); GOx and GOx (glucose oxidase); AOX and AOx (alcoholoxidase); COX and Cox (choline oxidase); HOx and HOx (hexose oxidase);SOx and SOx (sorbitol oxidase); PMS (phenazine methosulfate); NCBI(National Center for Biotechnology Information); ATCC (American TypeCulture Collection, Manassas, Va.); Geneart (Geneart, Regesburg,Germany); Invitrogen (Invitrogen, Inc., Carlsbad, Calif.); Stratagene(Stratagene, Inc., San Diego, Calif.); Biospringer (Biospringer, Inc.,Cedex, France); BASF (BASF, Mt. Olive, N.J.); and Sigma (Sigma-AldrichChemical Co., St. Louis, Mo.).

In the following Examples various growth media were used. In theseExamples, the TS medium was composed of 16 g Difco tryptone, 4 g Difcosoytone, 20 g caseine (hydrolysate) (Sigma), and 5 g K₂HPO₄ brought to 1liter. After autoclaving, 50% filtered sterilized glucose was added to afinal concentration of 1.5%. The “Production Medium” was composed of 2.4g citric acid*H₂O; 8.3 g yeast extract (Biospringer); 2.4 g (NH₄)₂SO₄;72.4 g MgSO₄*7H₂O; 0.1 g CaCl₂*2H₂O; 0.3 ml Mazu DF204 (BASF); 5 mlStreptomyces modified trace elements (1 liter stock solution contains:250 g citric acid* H₂O; 3.25 g FeSO₄*7H₂O; 5 g ZnSO₄*7H₂O; 5 gMnSO₄*H₂O; 0.25 g H₃BO₃); 10 g glucose, adjust volume to 1 liter and thepH was adjusted to 6.9, with NaOH. The R5 medium used was the standardR5 medium commonly used to grow Streptomyces.

Example 1 Construction of Strains Expressing Sorbitol Oxidase ofStreptomyces sp. H-7775 in Streptomyces lividans

In this Example, methods used to construct the strain that produced thesorbitol oxidase used in the development of the present invention aredescribed. The protein sequence (SEQ ID NO:2) of the sorbitol oxidasewas obtained from the published amino acid sequence (See e.g., Hiraga etal., Biosci. Biotechnol. Biochem., 62: 4347-353 [1998]). The signalsequence of the twin-arginine pathway of the Streptomyces ceolicolorSCO6772 gene (SEQ ID NO:15) was obtained from complete genome sequenceof Streptomyces coelicolor.

(SEQ ID NO: 15) MTEVSRRKLMKGAAVSGGALALPALGAPPATAAPAAGPEDLPGPAAA

The sorbitol oxidase was expressed in Streptomyces as a fusion proteinof the signal sequence of the SCO6772 protein (SEQ ID NO:15) andsorbitol oxidase (SEQ ID NO:2). A restriction site for NcoI wasintroduced at the 5′ end of DNA for cloning purposes, which resultedaddition of an amino acid glycine residue at position 2 (See, SEQ IDNO:16).

(SEQ ID NO: 16) MGTEVSRRKLMKGAAVSGGALALPALGAPPATAAPAAGPEDLPGPAAAMTPAEKNWAGNITFGAKRLCVPRSVRELRETVAASGAVRPLGTRHSFNTVADTSGDHVSLAGLPRVVDIDVPGRAVSLSAGLRFGEFAAELHARGLALANLGSLPHISVAGAVATGTHGSGVGNRSLAGAVRALSLVTADGETRTLRRTDEDFAGAVVSLGALGVVTSLELDLVPAFEVRQWVYEDLPEATLAARFDEVMSAAYSVSVFTDWRPGPVGQVWLKQRVGDEGARSVMPAEWLGARLADGPRHPVPGMPAGNCTAQQGVPGPWHERLPHFRMEFTPSNGDELQSEYFVARADAVAAYEALARLRDRIAPVLQVSELRTVAADDLWLSPAHGRDSVAFHFTWVPDAAAVAPVAGAIEEALAPFGARPHWGKVFSTAPEVLRTLYPRYADFEELV GRHDPEGTFRNAFLDRYFRR

A restriction site for BamHI was also introduced at the 3′ end of DNAfor cloning purposes. The codons of the fusion gene were optimized forexpression in Streptomyces lividans. DNA was synthesized by Geneart. TheDNA fragment spanning the two restriction sites (i.e., from NcoI toBamHI (SEQ ID NO:17)) was cloned into Streptomyces expression plasmidpKB105 (See, U.S. patent application Ser. No. 11/303,650, filed Dec. 16,2005, incorporated by reference in its entirety) which was cut withBamHI completely and NcoI partially.

(SEQ ID NO: 17) CCATGGGCACCGAGGTCTCCCGCCGCAAGCTGATGAAGGGCGCGGCGGTGTCGGGCGGCGCGCTGGCGCTGCCGGCCCTCGGCGCCCCGCCCGCCACCGCGGCGCCGGCCGCCGGCCCCGAGGACCTCCCGGGCCCCGCCGCCGCCATGACCCCGGCCGAGAAGAACTGGGCCGGCAACATCACCTTCGGCGCCAAGCGCCTGTGCGTCCCGCGCTCCGTCCGCGAGCTGCGCGAGACCGTGGCCGCCTCCGGCGCCGTGCGCCCGCTGGGCACCCGCCACTCGTTCAACACCGTCGCCGACACCTCCGGCGACCACGTGTCGCTGGCCGGCCTGCCGCGCGTCGTCGACATCGACGTCCCGGGCCGGGCCGTGTCCCTGTCCGCCGGCCTGCGCTTCGGCGAGTTCGCCGCCGAGCTGCACGCCCGCGGCCTGGCCCTGGCCAACCTGGGCTCCCTGCCGCACATCTCCGTGGCGGGCGCGGTCGCCACCGGCACCCACGGCTCCGGCGTCGGCAACCGCTCCCTGGCGGGCGCCGTCCGCGCCCTGTCCCTGGTCACCGCCGACGGCGAGACCCGCACCCTGCGCCGCACCGACGAGGACTTCGCCGGCGCCGTCGTGTCCCTGGGCGCCCTGGGCGTCGTCACCTCCCTGGAGCTGGACCTGGTCCCGGCCTTCGAGGTCCGCCAGTGGGTCTACGAGGACCTGCCCGAGGCCACCCTGGCCGCCCGCTTCGACGAGGTCATGTCCGCCGCCTACTCCGTGTCCGTGTTCACCGACTGGCGCCCGGGCCCGGTCGGCCAGGTCTGGCTGAAGCAGCGCGTCGGCGACGAGGGCGCCCGCTCCGTCATGCCGGCCGAGTGGCTGGGCGCCCGCCTGGCCGACGGCCCGCGCCACCCGGTCCCCGGCATGCCCGCCGGCAACTGCACCGCCCAGCAGGGCGTCCCGGGCCCGTGGCACGAGCGCCTGCCGCACTTCCGCATGGAGTTCACCCCGTCCAACGGCGACGAGCTGCAGTCCGAGTACTTCGTCGCCCGCGCGGACGCCGTCGCGGCCTACGAGGCGCTGGCCCGCCTGCGCGACCGCATCGCCCCGGTCCTGCAGGTCTCCGAGCTGCGCACCGTCGCCGCCGACGACCTGTGGCTGTCCCCGGCCCACGGCCGCGACTCCGTCGCCTTCCACTTCACCTGGGTCCCGGACGCCGCCGCCGTCGCCCCGGTCGCCGGCGCCATCGAGGAGGCCCTGGCCCCGTTCGGCGCCCGCCCGCACTGGGGCAAGGTGTTCTCCACCGCCCCCGAGGTCCTGCGCACCCTGTACCCGCGCTACGCCGACTTCGAGGAGCTGGTCGGCCGCCACGACCCCGAGGGCACCTTCCGCAACGCCTTCCTCGACCGCTACTTCCGCCGCTGAGGATCC

The expression plasmid (pKB105-TAT-SOx7775; FIG. 1; SEQ ID NO:29) wastransformed into Streptomyces lividans strain g3s3 (See, U.S. patentapplication Ser. No. 11/305,650, supra) and three transformants wereselected and grown in TS medium for 2-3 days in the presence of 50 ug/mlthiostrepton at 30° C. Cells were then transferred to a productionmedium free of antimicrobials and growth was continued for another threedays. Then, 1 ml of the culture was transferred to each of two culturetubes and the cells were removed by centrifugation under conditionssufficient to separate the cells from the supernatants. The supernatantsobtained from these two culture tubes were tested in enzyme activityassays.

pKB105 TAT-SOx7775 (9495 bps):

(SEQ ID NO: 29)ctagagatcgaacttcatgttcgagttcttgttcacgtagaagccggagatgtgagaggtgatctggaactgctcaccctcgttggtggtgacctggaggtaaagcaagtgacccttctggcggaggtggtaaggaacggggttccacggggagagagagatggccttgacggtcttgggaaggggagcttcggcgcgggggaggatggtcttgagagagggggagctagtaatgtcgtacttggacagggagtgctccttctccgacgcatcagccacctcagcggagatggcatcgtgcagagacagacccccggaggtaaccatgggcaccgaggtgtcccgccggaaactcatgaaaggcgccgcggtgtccggcggcgcgctggcgctgcccgcgctcggcgcaccgccggcgacggccgcgcccgccgcaggccccgaggacctccccggccccgcagcggcgatgaccccggccgagaagaactgggccggcaacatcaccttcggcgccaagcgcctgtgcgtcccgcgctccgtccgcgagctgcgcgagaccgtggccgcctccggcgccgtgcgcccgctgggcacccgccactcgttcaacaccgtcgccgacacctccggcgaccacgtgtcgctggccggcctgccgcgcgtcgtcgacatcgacgtcccgggccgggccgtgtccctgtccgccggcctgcgcttcggcgagttcgccgccgagctgcacgcccgcggcctggccctggccaacctgggctccctgccgcacatctccgtggcgggcgcggtcgccaccggcacccacggctccggcgtcggcaaccgctccctggcgggcgccgtccgcgccctgtccctggtcaccgccgacggcgagacccgcaccctgcgccgcaccgacgaggacttcgccggcgccgtcgtgtccctgggcgccctgggcgtcgtcacctccctggagctggacctggtcccggccttcgaggtccgccagtgggtctacgaggacctgcccgaggccaccctggccgcccgcttcgacgaggtcatgtccgccgcctactccgtgtccgtgttcaccgactggcgcccgggcccggtcggccaggtctggctgaagcagcgcgtcggcgacgagggcgcccgctccgtcatgccggccgagtggctgggcgcccgcctggccgacggcccgcgccacccggtccccggcatgcccgccggcaactgcaccgcccagcagggcgtcccgggcccgtggcacgagcgcctgccgcacttccgcatggagttcaccccgtccaacggcgacgagctgcagtccgagtacttcgtcgcccgcgcggacgccgtcgcggcctacgaggcgctggcccgcctgcgcgaccgcatcgccccggtcctgcaggtctccgagctgcgcaccgtcgccgccgacgacctgtggctgtccccggcccacggccgcgactccgtcgccttccacttcacctgggtcccggacgccgccgccgtcgccccggtcgccggcgccatcgaggaggccctggccccgttcggcgcccgcccgcactggggcaaggtgttctccaccgcccccgaggtcctgcgcaccctgtacccgcgctacgccgacttcgaggagctggtcggccgccacgaccccgagggcaccttccgcaacgccttcctcgaccgctacttccgccgctgaggatccgcgagcggatcggctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggacgtggcagtatcagcgccatgtccggcatatcccagccctccgcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcaagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccgaattcggggcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgacaccaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaaccgagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaacggaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggcccacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgagcgtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacgtaacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagcgcgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgcgatatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgacgatcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgctgccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttggatcgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgtcctaccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaatcaccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcatatgaagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgccctgagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgcgccatggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgccggcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggaccgtgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccggaccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccggcggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtgccaggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagccgggcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctgccactgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaagcgggcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcacccctccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtcgcggaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccgcgtgcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggtcctgggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatcaccgccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggaccggctcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcgactgatcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagaccggatcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgatcgtcctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgctcgacgagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccggcacggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgggaaggcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcggacggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctggcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacggcggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccgtgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggcaacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctcacggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccgacggcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctcgcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacgcggcgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgcaggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccagggacgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccgccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcgccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttccgcccgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggcagcgtcggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacggtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggccgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgcccgccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgcccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcggcagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggcgctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagcacaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccggtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgggccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct

Two oligos (SEQ ID NO:18 and SEQ ID NO:19) were obtained fromInvitrogen.

(SEQ ID NO: 18) GCGCTAGCCGGCCCCCCGGCACAGGCCATGACCCCGGCCGAGAAGAAC TGGG(SEQ ID NO: 19) CAGGAAACAGCTATGAC

The primers were used in PCR to amplify sorbitol oxidase gene and tofuse the sorbitol oxidase gene to the celA signal sequence. The PCRreaction mixture containing DNA, dNTPs, primer and 4% DMSO in 1× bufferwas heated to 98° C. for 4 minutes to denature the DNA templates.Herculase® II enzyme (Stratagene) was added to the tube and PCR reactionwas performed in 30 cycles of 98° C. for 30 seconds, 62° C. for 30seconds and 72° C. for 1 minute and 8 seconds. The final extension at72° C. was done for 5 minutes and the reaction was chilled to 4° C.

The resulting PCR fragment contained a portion of the celA signalsequence, the sorbitol oxidase gene, and a portion of vector sequencecontaining two restriction enzyme sites (SEQ ID NO:20).

(SEQ ID NO: 20) GCGCTAGCCGGCCCCCCGGCACAGGCCATGACCCCGGCCGAGAAGAACTGGGCCGGCAACATCACCTTCGGCGCCAAGCGCCTGTGCGTCCCGCGCTCCGTCCGCGAGCTGCGCGAGACCGTGGCCGCCTCCGGCGCCGTGCGCCCGCTGGGCACCCGCCACTCGTTCAACACCGTCGCCGACACCTCCGGCGACCACGTGTCGCTGGCCGGCCTGCCGCGCGTCGTCGACATCGACGTCCCGGGCCGGGCCGTGTCCCTGTCCGCCGGCCTGCGCTTCGGCGAGTTCGCCGCCGAGCTGCACGCCCGCGGCCTGGCCCTGGCCAACCTGGGCTCCCTGCCGCACATCTCCGTGGCGGGCGCGGTCGCCACCGGCACCCACGGCTCCGGCGTCGGCAACCGCTCCCTGGCGGGCGCCGTCCGCGCCCTGTCCCTGGTCACCGCCGACGGCGAGACCCGCACCCTGCGCCGCACCGACGAGGACTTCGCCGGCGCCGTCGTGTCCCTGGGCGCCCTGGGCGTCGTCACCTCCCTGGAGCTGGACCTGGTCCCGGCCTTCGAGGTCCGCCAGTGGGTCTACGAGGACCTGCCCGAGGCCACCCTGGCCGCCCGCTTCGACGAGGTCATGTCCGCCGCCTACTCCGTGTCCGTGTTCACCGACTGGCGCCCGGGCCCGGTCGGCCAGGTCTGGCTGAAGCAGCGCGTCGGCGACGAGGGCGCCCGCTCCGTCATGCCGGCCGAGTGGCTGGGCGCCCGCCTGGCCGACGGCCCGCGCCACCCGGTCCCCGGCATGCCCGCCGGCAACTGCACCGCCCAGCAGGGCGTCCCGGGCCCGTGGCACGAGCGCCTGCCGCACTTCCGCATGGAGTTCACCCCGTCCAACGGCGACGAGCTGCAGTCCGAGTACTTCGTCGCCCGCGCGGACGCCGTCGCGGCCTACGAGGCGCTGGCCCGCCTGCGCGACCGCATCGCCCCGGTCCTGCAGGTCTCCGAGCTGCGCACCGTCGCCGCCGACGACCTGTGGCTGTCCCCGGCCCACGGCCGCGACTCCGTCGCCTTCCACTTCACCTGGGTCCCGGACGCCGCCGCCGTCGCCCCGGTCGCCGGCGCCATCGAGGAGGCCCTGGCCCCGTTCGGCGCCCGCCCGCACTGGGGCAAGGTGTTCTCCACCGCCCCCGAGGTCCTGCGCACCCTGTACCCGCGCTACGCCGACTTCGAGGAGCTGGTCGGCCGCCACGACCCCGAGGGCACCTTCCGCAACGCCTTCCTCGACCGCTACTTCCGCCGCTGAGGATCCGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTG

The PCR fragment was digested with restriction enzymes NheI and BamHI toremove the vector sequence portion. The resulting fragment was thencloned to expression vector pKB105 to generate the plasmid“pKB105-CeIA-SOx7775” (See, FIG. 2; SEQ ID NO: 30). The expressionplasmid was transformed into Streptomyces lividans strain g3s3 and threetransformants were selected and grown in TS medium for 2-3 days in thepresence of 50 ug/ml thiostrepton at 30° C. Cells were then transferredto a production medium free of antibiotics and growth was continued foranother three days. Then, 1 ml sample was transferred to each of two newculture tubes and cells were removed and the enzyme was purified asdescribed in Example 5.

pKB105-celA-SOx7775 (9489bps): (SEQ ID NO: 30)ctagagatcgaacttcatgttcgagttcttgttcacgtagaagccggagatgtgagaggtgatctggaactgctcaccctcgttggtggtgacctggaggtaaagcaagtgacccttctggcggaggtggtaaggaacggggttccacggggagagagagatggccttgacggtcttgggaaggggagcttcggcgcgggggaggatggtcttgagagagggggagctagtaatgtcgtacttggacagggagtgctccttctccgacgcatcagccacctcagcggagatggcatcgtgcagagacagacccccggaggtaaccatgggctttgggagcgctcccatcgcgttgtgtccgcttcgcacgaggaggaacgctttgaaacgccttttggccctgctcgcgaccggcgtgtcgatcgtcggcctgactgcgctagccggccccccggcacaggccatgaccccggccgagaagaactgggccggcaacatcaccttcggcgccaagcgcctgtgcgtcccgcgctccgtccgcgagctgcgcgagaccgtggccgcctccggcgccgtgcgcccgctgggcacccgccactcgttcaacaccgtcgccgacacctccggcgaccacgtgtcgctggccggcctgccgcgcgtcgtcgacatcgacgtcccgggccgggccgtgtccctgtccgccggcctgcgcttcggcgagttcgccgccgagctgcacgcccgcggcctggccctggccaacctgggctccctgccgcacatctccgtggcgggcgcggtcgccaccggcacccacggctccggcgtcggcaaccgctccctggcgggcgccgtccgcgccctgtccctggtcaccgccgacggcgagacccgcaccctgcgccgcaccgacgaggacttcgccggcgccgtcgtgtccctgggcgccctgggcgtcgtcacctccctggagctggacctggtcccggccttcgaggtccgccagtgggtctacgaggacctgcccgaggccaccctggccgcccgcttcgacgaggtcatgtccgccgcctactccgtgtccgtgttcaccgactggcgcccgggcccggtcggccaggtctggctgaagcagcgcgtcggcgacgagggcgcccgctccgtcatgccggccgagtggctgggcgcccgcctggccgacggcccgcgccacccggtccccggcatgcccgccggcaactgcaccgcccagcagggcgtcccgggcccgtggcacgagcgcctgccgcacttccgcatggagttcaccccgtccaacggcgacgagctgcagtccgagtacttcgtcgcccgcgcggacgccgtcgcggcctacgaggcgctggcccgcctgcgcgaccgcatcgccccggtcctgcaggtctccgagctgcgcaccgtcgccgccgacgacctgtggctgtccccggcccacggccgcgactccgtcgccttccacttcacctgggtcccggacgccgccgccgtcgccccggtcgccggcgccatcgaggaggccctggccccgttcggcgcccgcccgcactggggcaaggtgttctccaccgcccccgaggtcctgcgcaccctgtacccgcgctacgccgacttcgaggagctggtcggccgccacgaccccgagggcaccttccgcaacgccttcctcgaccgctacttccgccgctgaggatccgcgagcggatcggctgaccggagcggggaggaggacgggcggccggcggaaaagtccgccggtccgctgaatcgctccccgggcacggacgtggcagtatcagcgccatgtccggcatatcccagccctccgcatgcaagcttggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcaagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcgccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccgaattcggggcatgcctgcaggagtggggaggcacgatggccgctttggtcgacctcaacgagacgatgaagccgtggaacgacaccaccccggcggccctgctggaccacacccggcactacaccttcgacgtctgatcatcactgacgaatcgaggtcgaggaaccgagcgtccgaggaacacaggcgcttatcggttggccgcgagattcctgtcgatcctctcgtgcagcgcgattccgagggaaacggaaacgttgagagactcggtctggctcatcatggggatggaaaccgaggcggaagacgcctcctcgaacaggtcggaaggcccacccttttcgctgccgaacagcaaggccagccgatccggattgtccccgagttccttcacggaaatgtcgccatccgccttgagcgtcatcagctgcataccgctgtcccgaatgaaggcgatggcctcctcgcgaccggagagaacgacgggaagggagaagacgtaacctcggctggccctttggagacgccggtccgcgatgctggtgatgtcactgtcgaccaggatgatccccgacgctccgagcgcgagcgacgtgcgtactatcgcgccgatgttcccgacgatcttcaccccgtcgagaacgacgacgtccccacgccggctcgcgatatcgccgaacctggccgggcgagggacgcgggcgatgccgaatgtcttggccttccgctcccccttgaacaactggttgacgatcgaggagtcgatgaggcggaccggtatgttctgccgcccgcacagatccagcaactcagatggaaaaggactgctgtcgctgccgtagacctcgatgaactccaccccggccgcgatgctgtgcatgaggggctcgacgtcctcgatcaacgttgtctttatgttggatcgcgacggcttggtgacatcgatgatccgctgcaccgcgggatcggacggatttgcgatggtgtccaactcagtcatggtcgtcctaccggctgctgtgttcagtgacgcgattcctggggtgtgacaccctacgcgacgatggcggatggctgccctgaccggcaatcaccaacgcaaggggaagtcgtcgctctctggcaaagctccccgctcttccccgtccgggacccgcgcggtcgatccccgcatatgaagtattcgccttgatcagtcccggtggacgcgccagcggcccgccggagcgacggactccccgacctcgatcgtgtcgccctgagcgtccacgtagacgttgcgtgagagcaggactgggccgccgccgaccgcaccgccctcaccaccgaccgcgaccgcgccatggccgccgccgacggcctggtcgccgccgccgcccgccggttcggcgcctgacccgaccaacccccgcggggcgccggcacttcgtgctggcgccccgcccccacccaccaggagaccgaccatgaccgacttcgacggacgcctgaccgaggggaccgtgaacctggtccaggaccccaacggcggtggctggtccgcccactgcgctgagcccggttgcgactgggccgacttcgccggaccgctcggcttccagggcctcgtggccatcgctcgccgacacacgcactgaccgcacgtcaaagccccgccggatacccggcggggctctcttcggccctccaagtcacaccagccccaaggggcgtcgggagtggcggagggaacctctggcccgattggtgccaggattcccaccagaccaaagagcaacgggccggacttcgcacctccgacccgtccgctcccagactcgcgccccttagccgggcgagacaggaacgttgctcgtgcccagagtacggagcgatgccgaggcattgccagatcggcccgccgggccccgctgccactgcgggaccgcaattgcccacacaccgggcaaacggccgcgtatctactgctcagaccgctgccggatggcagcgaagcgggcgatcgcgcgtgtgacgcgagatgccgcccgaggcaaaagcgaacaccttgggaaagaaacaacagagtttcccgcacccctccgacctgcggtttctccggacggggtggatggggagagcccgagaggcgacagcctctgggaagtaggaagcacgtcgcggaccgaggctgcccgactgcggaaagccgcccggtacagccgccgccggacgctgtggcggatcagcggggacgccgcgtgcaagggctgcggccgcgccctgatggaccctgcctccggcgtgatcgtcgcccagacggcggccggaacgtccgtggtcctgggcctgatgcggtgcgggcggatctggctctgcccggtctgcgccgccacgatccggcacaagcgggccgaggagatcaccgccgccgtggtcgagtggatcaagcgcggggggaccgcctacctggtcaccttcacggcccgccatgggcacacggaccggctcgcggacctcatggacgccctccagggcacccggaagacgccggacagcccccggcggccgggcgcctaccagcgactgatcacgggcggcacgtgggccggacgccgggccaaggacgggcaccgggccgccgaccgcgagggcatccgagaccggatcgggtacgtcggcatgatccgcgcgaccgaagtcaccgtggggcagatcaacggctggcacccgcacatccacgcgatcgtcctggtcggcggccggaccgagggggagcggtccgcgaagcagatcgtcgccaccttcgagccgaccggcgccgcgctcgacgagtggcaggggcactggcggtccgtgtggaccgccgccctgcgcaaggtcaaccccgccttcacgcccgacgaccggcacggcgtcgacttcaagcggctggagaccgagcgcgacgccaacgacctcgccgagtacatcgccaagacccaggacgggaaggcgcccgccctcgaactcgcccgcgccgacctcaagacggcgaccggcgggaacgtcgccccgttcgaactcctcggacggatcggggacctgaccggcggcatgaccgaggacgacgccgccggggtcggctcgctggagtggaacctctcgcgctggcacgagtacgagcgggcaacccggggacgccgggccatcgaatggacccgctacctgcggcagatgctcgggctcgacggcggcgacaccgaggccgacgacctcgatctgctcctggcggccgacgccgacggcggggagctgcgggccggggtcgccgtgaccgaggacggatggcacgcggtcacccgccgcgccctcgacctcgaggcgacccgggccgccgaaggcaaggacggcaacgaggattcggcggccgtgggcgaacgggtgcgggaggtcctggcgctggccgacgcggccgacacagtggtggtgctcacggcgggggaggtggccgaggcgtacgccgacatgctcgccgccctcgcccagcgccgcgaggaagcaactgcacgccgacggcgagagcaggacgacgaccaggacgacgacgccgacgaccgccaggagcgggccgcccggcacatcgcccggctcgcaagtgggcccacttcgcactaactcgctcccccccgccgtacgtcatcccggtgacgtacggcgggggtcggtgacgtacgcggcgacggcggccggggtcgaagccgcgggagtaatcctgggattactcgcccggggtcggccccgccggcacttcgtgcaggcggtacctcgcgcccgactcgcctcgctacgagacgtgccgcgtacggtcgtcggccatgagcaccaccacccccagggacgccgacggcgcgaagctctgcgcctggtgcggctcggagatcaagcaatccggcgtcggccggagccgggactactgccgccgctcctgccgccagcgggcgtacgaggcccggcgccagcgcgaggcgatcgtgtccgccgtggcgtcggcagtcgctcgccgagatacgtcacgtgacgaaatgcagcagccttccattccgtcacgtgacgaaactcgggccgcaggtcagagcacggttccgcccgctccggccctgccggacccccggctgcagctcgcccggccgccggtccccctgccgtccggcccgtcccagaggcagcgtcggcggctcctgcctcccccgcccggcgccgaccgggacccgcaaaccccttgatccgctgtcgggggtgatcactacggtgggtgccgaagtgatcacggggaggactgatgcaccaccaggaccgggaccaggaccaggcgttagcggcagtgctggccgcactgctcctggtcggcgggacgctgatcgtgcgggagctcctgggcctgtggcccgccgtggcggtcggcatggcgcccgccctcgccctctacggaggcccgcccgcggcccgccggatagccgtcgcggtcgaggtccgccggttccgccggcatcttgcccaccacgatcgggcagccggatgaccggccacgacggagccgcacggctgaccagctcgacggccgccacctcatcgcggcagcaggtgctccccagcaacccacgacggggctcagggtcgcctcacgcggctcagcaccgcgacggcgggggtacggcgctccgggaggctgacaggcgctcagacggccgcgtagggccgcgagtcccccacccctccccgctgccctgtcggcgagcacaacggcgatgcccgcagtcggcggagcaggcgccacgtaaaccgcccaccgatgccgcccccgtcgtgtgcgcgggccggtcggcggccgggccggagcggggcgaagacaggagcgtcggccgggccgtgggccgggccgcgcggcccgctcgcgggccgccttgatgacgtagggaaagttgtaccgcaaaaaacgcagcctgaactagttgcgatcct

Example 2 Identification of Genes with Sequence Identity/Similarity tothe Streptomyces sp. H7775 SOx Gene

In this Example, experiments conducted to identify genes with sequenceidentity/similarity with the Streptomyces sp. H-7775 sorbitol oxidasegene are described. In these experiments, homologues encoding polyoloxidases (e.g., sorbitol and xylitol oxidase) were identified using theprimary sequences of two functionally characterized polyol oxidases,namely Streptomyces sp. H-7775 sorbitol oxidase described herein and theStreptomyces sp. IKD472/FERM P-14339 xylitol oxidase as queries in BLASTanalyses on the non-redundant (nr) protein database of the NationalCenter for Biotechnology Information. Putative polyol oxidases wereidentified from different species of Streptomyces, Acidothermus,Arthrobacter, Brevibacterium, Frankia, Nocardia, Janibacter,Marinobacter, Burkholderia, Paracoccus, Chromabacterium, Thermobifida,Xanthomonas, Pseudomonas, Corynebacterium and Bacillus. In addition, theUniProtKB/TrEMBL/Swiss-Prot databases were also searched for genes whichfunction as sorbitol or xylitol oxidases. In addition to theStreptomyces sp. IKD472/FERM P-14339 xylitol oxidase, the following wereidentified as xylitol oxidases in these searches: XYOA STRCO (Q9ZBU1)from Streptomyces coelicolor, XYOA STRSI (Q9KX73) from Streptomyces sp.strain IKD472/FERM P-14339; Q2E2H5 ACICE from Acidothermuscellulolyticus 11 B; and Q82LCO STRAW from Streptomyces avermitilis.

In additional experiments, the genes encoding polyol oxidases (e.g.,sorbitol oxidase/xylito I oxidase) were obtained by utilizing sequencedgenomes to generate and screen genomic libraries of culturableorganisms. In additional embodiments, novel polyol oxidase genes areobtained from microbes that are unculturable by generating and screeningmetagenomic libraries.

One example of such a metagenome is the complex microbial consortiaderived from different soil sources. The microbiome metagenome database(JGI-DOE, USA) was searched for sequences with sequence identity to theSOx gene using the Function search: Genes in EC:1.1.3.41-Xylitoloxidase, resulted in the identification of five FAD/FMN-containingdehydrogenases and one probable xylitol oxidase. The following table(Table 1) provides various polyol oxidases identified in searches inwhich the Streptomyces sp. H7775 SOx gene was used to query the QuickBLAST P.

TABLE 1 Polyol Oxidases UniProt Accession Numbers Protein Name SourceSpecies Name P97011 Sorbitol oxidase [SOx] SEQ Streptomyces sp ID NO: 1Q9ZBU1 Probable xylitol oxidase (EC Streptomyces 1.1.3.41) SEQ ID NO: 9coelicolor Q2E2H5 Putative xylitol oxidase Acidothermus SEQ ID NO: 6cellulolyticus 11B Q82LC0 Putative xylitol oxidase Streptomyces SEQ IDNO: 8 avermitilis Q4NJL0 FAD linked oxidase Arthrobacter sp. FB24N-terminal SEQ ID NO: 12 Q9KX73 Xylitol oxidase (EC 1.1.3.41)Streptomyces sp. SEQ ID NO: 10 IKD472 Q412H8 FAD linked oxidase,Kineococcus N-terminal radiotolerans SRS30216

Example 3 Expression of the Streptomyces lividans Sorbitol Oxidase Genein Streptomyces lividans Strain g3s3

In this Example, experiments conducted to express the S. lividanssorbitol oxidase gene in S. lividans are described. Based on in silicoanalyses, two other Streptomyces species (S. coelicolor and S.avermitilis) were identified as having putative xylitol oxidase genes.The locus containing the Streptomyces coelicolor putative XOx gene(SCO6147) sequence was retrieved from the sequence database. Thecorresponding S. lividans gene was isolated using primers N2: 5′ctccagacgcgccgggtaggtttc (SEQ ID NO: 23), Is-n2 5′ctgctgcgccgaccactgaccc (SEQ ID NO:24) and genomic DNA from S. lividansas template. The protocol suggested by the manufacturer for GC-richtemplates was followed in a PCR reaction consisting of the Platinum PfxDNA polymerase with the Enhancer solution (Invitrogen). Two parallel PCRreactions were carried out. The first PCR reaction (reaction #1)utilized primers designated as us-sco1 5′ gcccatatgagcgacatcacggtcacc(SEQ ID NO:21) and Is-sco1 5′ ggatcctcagcccgcgagcacccc (SEQ ID NO:22).The previous PCR product was used as the template. The PCR reactionproduct was a 1.269 kb PCR fragment. The PCR conditions used were: 30cycles of denaturation at 94° C. for 55 seconds, annealing at 55° C. for55 seconds, followed by extension for 1-2 minutes at 68° C. Thepolymerase used was Platinum Pfx DNA polymerase plus enhancer solution(Invitrogen). The resulting PCR product was cloned directly in an E.coli vector PCR Blunt TOPO (Invitrogen) and the sequence of the clonedSOx gene verified by DNA sequencing using primers provided in the kit.

The second PCR reaction (reaction #2) utilized primers us-s1 5′gccatgggcgacatcacggtcaccaac (SEQ ID NO:25) and Is-s1 5′atggatcctcagcccgcgagcacccc (SEQ ID NO:26) were used in a PCR reactionusing the previous PCR product as template and the same conditions asabove.

The 1.268 kb PCR product was digested with NcoI and BamHI, and theresulting fragment was cloned directly into an NcoI (partial digest) andBamHI digested Streptomyces vector pKB105. The final construct was theexpression vector designated as pSMM-SOx (S. lividans) is shown in FIG.4. Five ul of plasmid DNA was used to transform Streptomyces g3s3protoplasts using methods known in the art.

The transformation reaction product was plated on R5 plates andincubated at 32° C. for 18 hours. A soft nutrient agar overlaycontaining thiostrepton at 50 ug/ml was then poured on the plates andthe plates were incubated for an additional 3 days. Single colonies wereused to inoculate a 250 ml flask with 20 ml TS-G media containingthiostrepton at 50 ug/ml. After 3 days of cultivation with shaking at30° C., 2 ml aliquots were used to inoculate a 250 ml flask containingthe Streptomyces Production Medium (See, above).

The cell pellets was collected by centrifugation, resuspended in bufferand disrupted, as described in Example 2. Table 2 shows the SOxactivities present in the cell-free extracts derived from the differentStreptomyces transformants. Twenty different transformants containingfour DNAs encoding the SOx gene were analyzed for SOx activity, asdescribed above. The results are provided in Table 2.

TABLE 2 SOx Activities of Twenty Different Streptomyces TransformantsSOx Activity (units/mg) Transformant SOx DNA # 17 DNA # 20 DNA # 22 DNA# 24 1 11.38 8.03 10.89 13.60 2 6.77 13.14 12.38 10.38 3 8.88 16.81 8.5517.03 4 9.37 12.32 16.30 18.28 5 7.34 11.68 0.02 18.14The activity of the SOx produced intracellularly by the E. coli hostcells was greater than the activity of the SOx fusion protein producedextracellularly by other host cells as described in Examples

Example 4 Expression of the Streptomyces sp. H-7775 in E. coli

In this Example, methods for construction of an expression system forthe expression of Streptomyces sp. H-7775 sorbitol oxidase (SOx) aredescribed. A synthetic gene (with neutral codons) encoding the H-7775SOx gene was used to express the sorbitol oxidase gene in E. coli strainBL21(DE3)pLysS. The expression vector pET 24a with the SOx gene wascloned as Nde1+BamH1 fragment. The resulting plasmid (See, FIG. 3) wastransformed into competent E. coli Top 0 cells (Invitrogen) according tothe manufacturers protocol. Kanamycin resistant transformants containingthe 1.2 kb SOx gene were identified by the direct colony PCR methodusing the TAQ Ready-to-go PCR beads (Amersham) using the PCR reactionconditions as suggested by the manufacturer. The following primers wereused: us-sco1 5′ gcccatatgagcgacatcacggtcacc (SEQ ID NO:21) and Is-sco15′ ggatcctcagcccgcgagcacccc (SEQ ID NO:22) in the PCR reaction.

The SOx positive transformants were cultivated and plasmid DNA wasisolated. Plasmid DNA containing the cloned SOx gene was then used tothe commercially available BL21(DE3)pLysS competent cells (cat. #200132; Stratagene) and following the protocol in the instructionmanual.

The entire transformation reaction was directly used to inoculate 250 mlflasks containing 25 ml LB, containing kanamycin (50 ug/ml) andchloramphenicol (34 ug/ml). The cultures were incubated overnight withshaking at 37° C. For each transformant, a 2 liter flask containing 500mls of LB containing kanamycin (50 ug/ml) and chloramphenicol (34 ug/ml)was then inoculated with 25 mls of the overnight culture and was furtherallowed to grow for 2 hours to reach approximately OD₆₀₀ values of0.4-0.6 (mid logarithmic phase). Then, IPTG at a final concentration of1 mM was added to the cultures. The cultures were further incubated foran additional 2 hours and then harvested by centrifugation. Theresulting pellets were resuspended in phosphate buffer and the cellswere passed through a French press for cell disruption/lysis. The SOxactivity was determined using 10ul cell lysate and 990 ul assay reagent(100 mM potassium phosphate, pH 7, 1% Sorbitol, 5 mM ABTS, 10 U/ml HRP)and incubated at 37° C. for 3 minutes.

Example 5 Purification of Sorbitol Oxidase

In this Example, methods used to purify sorbitol oxidase produced by S.lividans (SEQ ID NO:4) are described. Sorbitol oxidase from Streptomyceslividans is localized in mycelia. Thus, the enzyme was isolated by celllysis using a French press from cell-extract in 100 mM Kpi buffer,potassium phosphate, pH 7.0. The cell extract was heated to 50° C. forone hour, followed by centrifugation sufficient to remove the debris.The cell lysate supernatant was then mixed with ammonium sulfate to 32%saturation to precipitate the protein fraction containing sorbitoloxidase. The protein precipitate was kept at 4° C. overnight and thenwas separated from mother liquor by centrifugation 10,000 RPM usingSorvall centrifuge and SLA-1500 Sorvall rotor.

The protein precipitate was then washed with 32% saturated ammoniumsulfate solution. The washed protein precipitate was dissolved back inKpi buffer and the insoluble material was discarded. The solublefraction was dialyzed against 25 mM Kpi buffer, pH 7.0, overnight andthen further purified using affinity chromatography on the reactiveorange resin (Prometic). This partially purified sorbitol oxidasepreparation and fermentation broth cell lysate (EFT of 108 hrs) wereused as samples in experiments for biobleaching. The molecular weight ofthe enzyme was determined to be ˜45,000 Da by SDS-PAGE gelelectrophoresis. The prosthetic group is a covalently bound FAD (1 molof FAD to 1 mol of SOx).

Example 6 Stability and Bleaching Performance of SOx in HDL Laundry WashConditions

In this Example, methods to determine the stability and bleachingperformance of SOx (produced as described above) in AATCC liquiddetergent laundry wash conditions are described. In these experiments,AATCC standard detergent (American Association of Textile Chemists andColorists Heavy Duty Liquid Detergent Version 2003 without brightener;key components include linear alkane sulfonate, alcohol ethoxylate,propanediol, citric acid, fatty acid, castic soda and water;Testfabrics) was used.

Three bleachable cotton swatches with juice (STC CFT CS-15), wine (STCCFT CS-3), and tea (STC CFT BC-3) were used. The swatches were cut into15 mm circles with a textile punch (Model B equipped with a ⅝″ diecutter; Model 93046; NAEF).

Single swatch disks were placed into each well of a 24-well microplate(Costar 3526). One (1) ml of washing solution pH 8, containing perliter, 1.5 ml AATCC HDL detergent, 75-100 mM sorbitol, 6 gpg hardness(diluted from stock 15000 gpg hardness solution containing 1.735 Mcalcium chloride and 0.67 M magnesium chloride), and 0.05% TAED(tetraacetylethylenediamine, Fluka) was added to each well. Five tofifty (5-50 ul) microliters of partially purified sorbitol oxidase orsorbitol oxidase obtain from a late fermentation run (108 hr EFT“effective fermentation time”) produced as described in Examples 1 and2, were added with a positive displacement pipette to 3-8 wells in onecolumn. The control wells contained no enzyme. The microplate wascovered with a plastic lid and aluminum foil and incubated at 37° C.with 100 rpm gentle rotation for 14 hr. The plates were then removedfrom the shaker and tested for the presence of hydrogen peroxide withperoxide test strips (Baker).

One hundred microliters (100 ul) of 0.1 mM sodium carbonate were addedto each well to elevate the pH to 10. The microplates were incubatedwith rotation for another 90 min and the supernatants removed byaspiration. Each well was washed three (3) times with 1.5 ml Dulbecco'sPBS, pH 7.3 and three (3) times with 1.5 ml distilled water. Each diskwas removed from its well and dried overnight between sheets of papertowels and not exposed to direct light. The disks were inspectedvisually and then analyzed with a Reflectometer CR-200 (Minolta)calibrated on a standard white tile. The average L values werecalculated as was the percent soil release (% SR=100% X (Finalreflectance-Initial reflectance)/(Reflectance of a whitestandard-Initial reflectance). Graphs showing the results of theseexperiments are provided in FIGS. 5-8.

The results confirmed that sorbitol oxidase is stable in a typicalliquid detergent system and is able to produce effective concentrationof hydrogen peroxide in presence of its substrate sorbitol mixed withdetergent and available atmospheric oxygen. In addition, the sorbitoloxidase generated hydrogen peroxide in presence of a bleach booster(i.e., TAED) was able to help bleach typical colored stains such asblueberry, tea and wine (See, FIGS. 5-8).

Example 7 Substrate Range Study of Sorbitol Oxidase

Sorbitol oxidase obtained using the methods described in Example 1, wastested for finding its activity with various polyol substrates. Allsubstrates used in the assay were 50 mM in 10 mM phosphate buffer pH7.0. The relative activity using sorbitol as (++++++=100%) is shownbelow in Table 3. Although the activity was very high for bothsubstrates, it was noted that the SCO6147 SOx transformed in S. lividansexhibited 2.1 times more activity on D-sorbitol than on D-xylitol.

In addition to these substrates, it is contemplated that othersubstrates, including, but not limited to glycerol, will find use in thepresent invention.

TABLE 3 Relative POx activities for corresponding substrates CompoundRelative Activity (+, 0) D-Sorbitol ++++++ D-Xylitol ++++ D-Mannitol +++D-Ribitol + Myo-Inositol + Glycerol + 1,3-propanediol +/21,2-propanediol +/2 Propylene glycol 0 Ethylene glycol 0

Example 8 Combined Enzyme System: Increased Hydrogen Peroxide ProductionUsing SOx+GOx (Glucose Oxidase) and SOx+HOx (Hexose Oxidase)

The activity of a combination of POx enzymes was tested. The enzymesthat were tested in combination were SOx with GOx and SOx with HOx. TheSOx used in the experiments was recombinant SOx purified from S.lividans strain transformant # 4 (SEQ ID NO:4), GOx is commerciallyavailable from Sigma (G7141) and HOx was prepared as described in WO01/38544. HOx is available from Danisco A/S as DairyHOx™.

SOx, HOx and GOx have been discussed above, and can be summarized asfollows.

Hexose oxidase (EC. 1.1.3.5) is a homo dimeric flavo enzyme containing 2covalently bound FAD groups per molecule. The molecular weight isapprox. 130 kDa. The enzyme catalyses the oxidation of D-hexose sugarsincluding: glucose, galactose, lactose, maltose, malto-triose andcellobiose. The sugars are converted into their corresponding lactones,which undergo spontaneous hydrolysis to the corresponding acids inaqueous environments. Upon re-oxidation of the reduced flavin group bymolecular oxygen, H₂O₂ is produced.

Sorbitol oxidase is a monomeric flavo enzyme containing one flavin groupof unknown nature, bound covalently, probably to a histidine residue inthe holoenzyme. The molecular weight of the enzyme is approximately 45kDa. The enzyme catalyses the oxidation of several polyols including:D-sorbitol, D-xylitol, D-glucose, D-mannitol, D-arabitol, glycerol,inositol, 1,2-propanediol, 1,3-butanediol, and 1,4-butanediol. Uponoxidation of the polyols the corresponding sugars are generated and uponcontact with molecular oxygen the flavin is re-oxidized and H₂O₂ isproduced.

Glucose oxidase (EC. 1.1.3.4) is a dimeric protein with a molecularweight of about 160 kDa containing 1 non-covalently, but tightlyassociated flavin group per monomer. The enzyme catalyses the oxidationsof D-glucose and to a lesser extend 2-deoxy-D-glucose, D-mannose andD-fructose. The sugars are converted into their corresponding lactones,which undergo spontaneous hydrolysis to the corresponding acids inaqueous environments. Upon re-oxidation of the reduced flavin group bymolecular oxygen, H₂O₂ is produced.

Activity assays of SOx, HOx or GOx activity were performed in microtiterplates (300 μl).

The amount of enzyme in each combination was given as unit amount,wherein a unit of HOx is the amount of enzyme that produced 1 umolH2O2/min when substrates are in excess; a unit of GOx is the amount ofenzyme that produced 1 umol H2O2/min when substrates are in excess; anda unit of SOx is the amount of enzyme that produced 1 umol H2O2/min whensubstrates are in excess. In the present example, only SOx had excesssubstrate. The generated sugar would be the limiting factor for thesecondary enzyme. As noted above the activity is given as a percentagevalue of SOx catalyzing D-sorbitol when both D-sorbitol and oxygen is inexcess.

The commonly used horse radish peroxidase dye substrate ABTS wasincorporated into an assay, measuring the production of H₂O₂ produced byHOx or GOx respectively. ABTS serves as a chromogenic substrate forperoxidase. Peroxidase in combination with H₂O₂ facilitates the electrontransport from the chromogenic dye, which is oxidized to an intenselygreen/blue compound.

An assay mixture contained 266 μl sorbitol (Sigma P-5504, 0.055 M in 0.1M sodium phosphate buffer, pH 6.3), 11.6 μl2,2′-Azino-bis(3-ethylbenzothiozoline-6-Sulfonic acid) (ABTS) (SigmaA-9941, 5 mg/ml aqueous solution), 11.6 μl peroxidase (POD) (SigmaP-6782, 0.1 mg/ml in 0.1 M sodium phosphate buffer, pH 6.3) and 10 μlenzyme (SOx or HOx) aqueous solution.

The incubation was started by the addition of glucose at 25° C. Theabsorbance was monitored at 405 nm in an ELISA reader. A standard curve,based on varying concentrations of H₂O₂, was used for calculation ofenzyme activity according to the definition above.

Initial velocities were measured over 5 minutes in 300 uL ABTS assay (asdescribed previously). The production of rate of hydrogen peroxideproduction was extrapolated from a standard curve. The measured activityis given as a percentage value. 100% is defined as the rate of hydrogenperoxide production by sorbitol oxidase alone, when the substratesD-sorbitol and oxygen is in excess (Linear velocity curves).

FIG. 9 (A-B) shows the initial velocity of H₂0₂ production using thecompositions with between 1× and 3000× excess of the oxidoreductases(glucose oxidase & hexose oxidase) compared to the polyol oxidase(sorbitol oxidase) measured over 5 minutes in 300 ul ABTS assay. Adramatic synergy was seen with both glucose oxidase and hexose oxidase,with up to 250-300% increase in hydrogen peroxide production rate seen.Dosages greater than 1×, such as at least 2× of the oxidoreductasecompared to the polyol oxidase resulted in increased hydrogen peroxideproduction.

Example 9 Construction of Streptomyces lividans & Bacillus subtilisStrains Expressing Polyol Oxidases (POx)

Table 4 shows the elements of polynucleotide constructs that weregenerated to express POx enzymes derived from A. cellulolyticus,Arthrobacter sp, and Streptomyces H-7775 in Bacillus and/or Streptomyceshost cells.

TABLE 4 Expression Polyol Oxidase (POx) Genes N-terminal Constructs(sorbitol/xylitol oxidases) fusion to POx Expression Host pSMM-ES2Acidothermus cellulolyticus 11B signal peptide Streptomyces Q2E2H5(synthetic gene); SCO7637 lividans amino acid sequence SEQ ID NO: 6 SEQID NO: 31 pSMM- Acidothermus cellulolyticus 11B none StreptomycesPOx(intra) Q2E2H5 (synthetic gene) lividans amino acid sequence SEQ IDNO: 6 pSM-ES3 Arthrobacter sp. FB 24 Signal peptide Streptomyces(Q4NJLO) (synthetic gene) SCO0624 lividans amino acid sequence SEQ IDNO: 12 SEQ ID NO: 39 pSM CG-SOx Streptomyces H-7775 Cgt signal Bacillussubtilis (synthetic gene) sequence. amino acid sequence SEQ ID NO: 2 SEQID NO: 36 pSM CG-SOx Acidothermus cellulolyticus 11B Cgt signal Bacillussubtilis (ES2 Acid.) Q2E2H5 (synthetic gene) sequence. amino acidsequence SEQ ID NO: 6 SEQ ID NO: 36

For extracellular expression of POx, the following signal sequences werefused to the mature form of the POx enzyme as described herein.

MGFGSAPIALCPLRTRRNALKRLLALLATGVSIVGLTALAGPPAQA (SEQ ID NO:31) secretedendoglucanase from Streptomyces coelicolor SCO7363MHEPHLDRRLFLKGTAVTGAALALGATAAPTASA (SEQ ID NO:39) possible secretedprotein Streptomyces coelicolor SCO0624MKKFLKSTAALALGLSLTFGLFSPAQA (SEQ ID NO:36) amino acid signal sequence ofthe Bacillus circulans cyclomaltodextrin glucanotransferase (cgt)precursor (accession P43379).

Example 10 Construction of Streptomyces lividans & Bacillus subtilisStrains Expressing Polyol Oxidase (POx) from Acidothermus cellulolyticus11B

In this example, experiments were conducted to functionally characterizethe putative polyol oxidase (xylitol oxidase) Q2E2H5 from Acidothermuscellulolyticus 11B. The putative POx protein was expressed in twodifferent host organisms (Streptomyces lividans and Bacillus subtilis).The POx protein sequence was retrieved from the database (SEQ ID NO:6).A synthetic polynucleotide encoding the Acidothermus cellulolyticus 118Q2E2H5 POx protein was obtained from GeneArt (Germany).

The Acidothermus POx synthetic gene was designed with a truncated signalsequence ALAGPPAQA (SEQ ID NO:32) from a secreted endoglucanase fromStreptomyces coelicolor SCO7363. Acidothermus POx was expressed inStreptomyces as a fusion protein (SEQ ID NO:35), resulting in theN-terminal extension MGFGSAPIALCPLRTRRNALKRLLALLATGVSIVGLTALAGPPAQA (SEQID NO:31) (from SCO7363 signal sequence) fused to the mature POxsequence (SEQ ID NO:6). The amino acid sequence encoded by the syntheticAcidothermus POx gene (SEQ ID NO:33) is as follows.

(SEQ ID NO: 33) ALAGPPAQAMDGGKRCRDGTPQPPAPSEQVTPSAAASLRAAYDVEVSAPRLRNWAGNIAFRPRRYVQPRDLDELVEIIRVSDQVRVLGTGHSFNPIADTTGTLISLDHLPREVRVMPGRTAVSAGTRYGDLAFPLHEAGWALANVGSLPHISIAGACATATHGSGDRNGCLATAVAGMTGVDGTCRVFHLTAESPEFPGAVVHLGALGAVTEIELVTEPTFTVRQWVYEDAPLDNVFADLDDVTSAAYSVSIFTTWDPPTARQIWLKERVAAGRPDPPARRWGGRLAERDHNPVPGMPPENCTPQLGRIGPWHERLPHFRLDVTPSAGDELQSEYFVPRAAAVEAYRALRHIGSRIAPVLQISEIRTVAADELWLSPAYHRPSVAFHFTWIADEEAVRPVVSEVERALAPLQPRPHWGKLFTMDPAVVRAAYPRFDDFVALAERYDPE GKFQNDFLRRFFAGThe polynucleotide sequence of the synthetic Acidothermus POx gene (SEQID NO:34) is as follows.

(SEQ ID NO: 34) GCGCTAGCGGGCCCGCCGGCCCAGGCC ATGGATGGCGGCAAGCGCTGCCGCGACGGCACCCCGCAGCCGCCGGCCCCGTCCGAGCAGGTCACCCCGTCGGCCGCCGCCTCCCTGCGGGCCGCCTACGACGTGGAGGTCTCCGCCCCGCGCCTGCGCAACTGGGCCGGCAACATCGCCTTCCGCCCGCGCCGCTACGTCCAGCCGCGCGACCTCGACGAGCTGGTCGAGATCATCCGGGTCTCCGACCAGGTCCGCGTCCTGGGCACCGGCCACTCCTTCAACCCCATCGCCGACACCACCGGCACCCTGATCTCCCTGGACCACCTGCCGCGCGAGGTCCGCGTCATGCCGGGCCGCACCGCGGTCTCCGCCGGCACCCGCTACGGCGACCTGGCCTTCCCGCTGCACGAGGCCGGCTGGGCCCTGGCCAACGTCGGCTCCCTGCCGCACATCTCCATCGCCGGCGCCTGCGCCACGGCCACCCACGGCTCCGGCGACCGCAACGGCTGCCTGGCCACCGCCGTCGCCGGCATGACCGGCGTCGACGGCACCTGCCGCGTGTTCCACCTGACCGCCGAGTCCCCCGAGTTCCCGGGCGCCGTCGTCCACCTGGGCGCCCTGGGCGCCGTCACCGAGATCGAGCTGGTCACCGAGCCGACCTTCACCGTCCGCCAGTGGGTCTACGAGGACGCCCCGCTGGACAACGTGTTCGCCGACCTGGACGACGTCACCTCCGCCGCCTACTCGGTCTCCATCTTCACCACCTGGGACCCGCCGACCGCCCGGCAGATCTGGCTGAAGGAGCGCGTCGCCGCCGGCCGCCCGGACCCGCCGGCCCGCCGCTGGGGCGGCCGCCTCGCCGAGCGCGACCACAACCCCGTCCCGGGGATGCCGCCCGAGAACTGCACCCCCCAGCTGGGCCGCATCGGCCCGTGGCACGAGCGCCTGCCGCACTTCCGCCTGGACGTCACCCCCTCCGCGGGCGACGAGCTGCAGTCCGAGTACTTCGTCCCGCGCGCCGCCGCCGTCGAGGCCTACCGCGCCCTGCGCCACATCGGCTCCCGCATCGCCCCGGTCCTGCAGATCTCCGAGATCCGCACCGTCGCCGCCGACGAGCTGTGGCTGTCCCCGGCCTACCACCGCCCGTCCGTCGCCTTCCACTTCACCTGGATCGCCGACGAGGAGGCCGTCCGCCCGGTGGTCTCCGAGGTCGAGCGCGCCCTGGCCCCGCTGCAGCCGCGCCCGCACTGGGGCAAGCTGTTCACGATGGACCCGGCCGTCGTCCGCGCCGCCTACCCGCGCTTCGACGACTTCGTCGCCCTGGCCGAGCGCTACGACCCCGAGGGCAAGTTCCAGAACGACTTCCTGCGCCGCTTCTTCGCCGGCTAAGGATCC.

A restriction site for nhe1 (GCTAGC) was introduced at the 5′ end of thesynthetic gene to allow fusion to the truncated SCO7363 endoglucanasesignal sequence. The BamH1 (GGATCC) restriction site was added at the 3′end of the gene. Restriction digestion with nhe1 plus Bam H1 resulted inthe DNA fragment which was ligated with the nhe1/BamH1 cut Streptomycesexpression plasmid pKB 105. The cloning resulted in the expressionconstruct pSMM-ES2 with a full length signal peptide for expression ofsecreted POx proteins in Streptomyces lividans (SEQ ID NO: 35).

(SEQ ID NO: 35) MGFGSAPIALCPLRTRRNALKRLLALLATGVSIVGLTALAGPPAQAMDGGKRCRDGTPQPPAPSEQVTPSAAASLRAAYDVEVSAPRLRNWAGNIAFRPRRYVQPRDLDELVEIIRVSDQVRVLGTGHSFNPIADTTGTLISLDHLPREVRVMPGRTAVSAGTRYGDLAFPLHEAGWALANVGSLPHISIAGACATATHGSGDRNGCLATAVAGMTGVDGTCRVFHLTAESPEFPGAVVHLGALGAVTEIELVTEPTFTVRQWVYEDAPLDNVFADLDDVTSAAYSVSIFTTWDPPTARQIWLKERVAAGRPDPPARRWGGRLAERDHNPVPGMPPENCTPOLGRIGPWHERLPHFRLDVTPSAGDELOSEYFVPRAAAVEAYRALRHIGSRIAPVLQISEIRTVAADELWLSPAYHRPSVAFHFTWIADEEAVRPVVSEVERALAPLQPRPHWGKLFTMDPAVVRAAYPRFDDFVALAERYDPEGKFQNDFLRRFFAG

The DNA fragment encoding the mature POx protein from Acidothermus (SEQID NO:6) (without a signal peptide) was also generated (SEQ ID NO:42).

(SEQ ID NO: 42) CCATGGATGGCGGCAAGCGCTGCCGCGACGGCACCCCGCAGCCGCCGGCCCCGTCCGAGCAGGTCACCCCGTCGGCCGCCGCCTCCCTGCGGGCCGCCTACGACGTGGAGGTCTCCGCCCCGCGCCTGCGCAACTGGGCCGGCAACATCGCCTTCCGCCCGCGCCGCTACGTCCAGCCGCGCGACCTCGACGAGCTGGTCGAGATCATCCGGGTCTCCGACCAGGTCCGCGTCCTGGGCACCGGCCACTCCTTCAACCCCATCGCCGACACCACCGGCACCCTGATCTCCCTGGACCACCTGCCGCGCGAGGTCCGCGTCATGCCGGGCCGCACCGCGGTCTCCGCCGGCACCCGCTACGGCGACCTGGCCTTCCCGCTGCACGAGGCCGGCTGGGCCCTGGCCAACGTCGGCTCCCTGCCGCACATCTCCATCGCCGGCGCCTGCGCCACGGCCACCCACGGCTCCGGCGACCGCAACGGCTGCCTGGCCACCGCCGTCGCCGGCATGACCGGCGTCGACGGCACCTGCCGCGTGTTCCACCTGACCGCCGAGTCCCCCGAGTTCCCGGGCGCCGTCGTCCACCTGGGCGCCCTGGGCGCCGTCACCGAGATCGAGCTGGTCACCGAGCCGACCTTCACCGTCCGCCAGTGGGTCTACGAGGACGCCCCGCTGGACAACGTGTTCGCCGACCTGGACGACGTCACCTCCGCCGCCTACTCGGTCTCCATCTTCACCACCTGGGACCCGCCGACCGCCCGGCAGATCTGGCTGAAGGAGCGCGTCGCCGCCGGCCGCCCGGACCCGCCGGCCCGCCGCTGGGGCGGCCGCCTCGCCGAGCGCGACCACAACCCCGTCCCGGGGATGCCGCCCGAGAACTGCACCCCCCAGCTGGGCCGCATCGGCCCGTGGCACGAGCGCCTGCCGCACTTCCGCCTGGACGTCACCCCCTCCGCGGGCGACGAGCTGCAGTCCGAGTACTTCGTCCCGCGCGCCGCCGCCGTCGAGGCCTACCGCGCCCTGCGCCACATCGGCTCCCGCATCGCCCCGGTCCTGCAGATCTCCGAGATCCGCACCGTCGCCGCCGACGAGCTGTGGCTGTCCCCGGCCTACCACCGCCCGTCCGTCGCCTTCCACTTCACCTGGATCGCCGACGAGGAGGCCGTCCGCCCGGTGGTCTCCGAGGTCGAGCGCGCCCTGGCCCCGCTGCAGCCGCGCCCGCACTGGGGCAAGCTGTTCACGATGGACCCGGCCGTCGTCCGCGCCGCCTACCCGCGCTTCGACGACTTCGTCGCCCTGGCCGAGCGCTACGACCCCGAGGGCAAGTTCCAGAACGACTTCCTGCGCCGCTTCTTCGCCGGCTAAGGATCC.

Restriction digestion with Nco1 and BarnH1 resulted in a DNA fragmentwhich was ligated to the Nco1 partial digest plus BamH1 cut pKB 105vector. Cloning resulted in the expression construct pSMM-POx(intra),for the intracellular expression of the mature Acidothermus POx protein.

The expression plasmids pSMM-ES2 and pSMM-POx(intra) were transformedinto Streptomyces lividans strain g3s3 and 10 transformants each wereselected and grown in TS medium for 2-3 days in the presence of 50 ug/mlthiostrepton at 30° C. Cells were then transferred to a productionmedium free of antimicrobials and growth was continued for another threedays. Then, 1 ml cultures was collected and centrifuged under conditionssufficient to separate the cells from the supernatants. The supernatantsand cell pellets obtained were tested in enzyme activity assays.

The Nco1/BamH1 digested DNA fragment was also used to construct theexpression plasmid pSM CG-SOx (ES2 Acid.). The Bacillus subtilis vectorpCG (Danisco A/S) was digested with Nco1/BamH1 and ligated to the POxDNA fragment. The resulting fusion protein (SEQ ID NO:37) comprises themature POx gene to the 27 amino acids signal peptideMKKFLKSTAALALGLSLTFGLFSPAQA (SEQ ID NO: 36) of the Bacillus circulanscyclomaltodextrin glucanotransferase precursor (accession P43379).

(SEQ ID NO: 37) MKKFLKSTAALALGLSLTFGLFSPAQAMDGGKRCRDGTPQPPAPSEQVTPSAAASLRAAYDVEVSAPRLRNWAGNIAFRPRRYVQPRDLDELVEIIRVSDQVRVLGTGHSFNPIADTTGTLISLDHLPREVRVMPGRTAVSAGTRYGDLAFPLHEAGWALANVGSLPHISIAGACATATHGSGDRNGCLATAVAGMTGVDGTCRVFHLTAESPEFPGAVVHLGALGAVTEIELVTEPTFTVRQWVYEDAPLDNVFADLDDVTSAAYSVSIFTTWDPPTARQIWLKERVAAGRPDPPARRWGGRLAERDHNPVPGMPPENCTPQLGRIGPWHERLPHFRLDVTPSAGDELQSEYFVPRAAAVEAYRALRHIGSRIAPVLQISEIRTVAADELWLSPAYHRPSVAFHFTWIADEEAVRPVVSEVERALAPLQPRPHWGKLFTMDPAVVRAAYPRFDDFVALAERYDPEGKFQNDFLRRFFAG.

The pSM CG-SOx (ES2 Acid.) and pSM CG-SOx plasmids were used fortransformation of the Bacillus subtilis OS21 strain. Transformants wereisolated and grown in 5 ml LB media containing 50 mg/li Kanamycin. Thecultures were cultivated at 30° C. with shaking at 180 rpm for 24 hours.SOx activities were determined from culture supernatants of host cellssecreting the SOx and cell lysates of host cells that produced SOxintracellularly.

The data obtained from the experiments showed that the SOx produced fromhost cells that had been transformed with a polynucleotide encoding themature form of the SOx enzyme (i.e. lacking the sequence encoding thesignal peptide) had greater SOx activity than the SOx produced by hostcells that had been transformed with a polynucleotide encoding thefusion SOx protein (i.e. a SOx fusion protein comprising the mature formoperably linked to a signal peptide).

Example 13 Construction of Streptomyces lividans Strains ExpressingPolyol Oxidase (POx) from Arthrobacter sp. FB 24

In this example, experiments were conducted to functionally characterizethe putative polyol oxidase (FAD linked oxidase) (Q4NJLO; SEQ ID NO:11)from Arthrobacter sp. FB 24 are described. The putative POx protein wasexpressed in Streptomyces lividans. The POx protein sequence wasretrieved from the database. Synthetic gene encoding the Arthrobactersp. FB 24 (Q4NJLO) POx protein was obtained from Generate (Germany).

The Arthrobacter sp. FB 24 POx synthetic gene was designed with anN-terminal extension MHEPHLDRRLFLKGTAVTGAALALGATAAPTASA (SEQ ID NO:39)derived from a possible secreted Streptomyces coelicolor A3(2) proteinSCO0624 resulting in the protein sequence below (SEQ ID NO:40). Twoglycine residues were inserted after the initiator methionine of thesignal sequence and before the start methionine of the mature POxprotein.

(SEQ ID NO: 40) MGHEPHLDRRLFLKGTAVTGAALALGATAAPTASA GMRTVSELPGLSGSTGAGSSAPELNWAGNYRYTAASIHRPRTLEEVQEVVAGASKIRALGSRHSFNAIADSPGSLVSLEDLDPGIRIDAATRTVTVSGGTRYGTLAEQLESAGFALSNLASLPHISVAGAIATATHGSGDANGNLATSVAALELVAADGTVHRLNRGSSPGFDGAVVGLGALGVVTKVTLDIEPTFTVRQDVFEALPWDTVLGNFDAVTSSAYSVSLFTDWSGDDVAQAWLKSRLSGSAASSDAGSTLAGEAFAAGTFFGGTRAGVARHPLPGVSAENCTEQLGVPGSWSERLAHFRMAFTPSSGEELQSEFFVRREHAVAAIGELRALSDRITPLLLVSEIRTVAADKLWLSTAYGQDSVGFHFTWKQRQDEVEKVLPVMEEALAPFNARPHWGKLFHAGADAVAELYPRFSDFKDLAERMDPEQKFRNEFLARKVFGN

The two glycine residues resulted from incorporating restriction sitesNco1 and sph1. as shown in the synthetic gene sequence below (SEQ IDNO:41). The synthetic POx gene was cut with the restriction enzymes Nco1and BamH1 and the resulting DNA fragment was ligated to the Nco1 partialand BamH1 digested Streptomyces vector pKB105. This resulted in theexpression vector pSM-ES3. This plasmid was transformed intoStreptomyces lividans strain g3s3 and 10 transformants were selected andgrown in TS medium for 2-3 days in the presence of 50 ug/ml thiostreptonat 30° C. Cells were then transferred to a production medium free ofantimicrobials and growth was continued for another three days. Then, 1ml cultures was collected and centrifuged under conditions sufficient toseparate the cells from the supernatants. The supernatants and cellpellets obtained were tested in enzyme activity assays.

(SEQ ID NO: 41) ACC ATGGGCCACGAGCCGCACCTGGACCGCCGCCTGTTCCTGAAGGGCACCGCCGTCACCGGCGCCGCCCTGGCCCTGGGCGCCACCGCCGCCCCGACCGCCTCCGCCG GCATGCGCACGGTCTCCGAGCTGCCGGGCCTGTCGGGCTCCACCGGCGCCGGCTCCTCCGCCCCCGAGCTGAACTGGGCCGGCAACTACCGCTACACCGCCGCCTCCATCCACCGCCCGCGCACCCTCGAGGAGGTCCAGGAGGTCGTCGCGGGCGCCTCCAAGATCCGCGCCCTGGGCTCCCGCCACTCCTTCAACGCCATCGCCGACTCCCCGGGCAGCCTGGTCTCCCTCGAGGACCTGGACCCGGGCATCCGCATCGACGCCGCCACCCGCACCGTCACGGTCTCGGGCGGCACGCGCTACGGCACCCTGGCCGAGCAGCTCGAGTCCGCCGGCTTCGCCCTGTCCAACCTGGCCTCCCTGCCGCACATCTCCGTCGCCGGCGCCATCGCCACCGCCACCCACGGCTCCGGCGACGCCAACGGCAACCTGGCCACCTCCGTCGCCGCCCTCGAGCTGGTCGCGGCCGACGGCACCGTCCACCGCCTGAACCGCGGCTCCTCCCCGGGCTTCGACGGCGCGGTCGTCGGCCTGGGCGCCCTGGGCGTCGTCACCAAGGTCACCCTGGACATCGAGCCGACCTTCACCGTCCGCCAGGACGTGTTCGAGGCCCTGCCGTGGGACACCGTCCTGGGCAACTTCGACGCCGTCACCTCCTCCGCCTACTCCGTGTCCCTGTTCACCGACTGGTCCGGCGACGACGTCGCCCAGGCCTGGCTGAAGTCCCGCCTGTCCGGCTCCGCCGCCTCCTCCGACGCCGGCTCCACCCTGGCCGGCGAGGCCTTCGCCGCCGGCACCTTCTTCGGCGGCACCCGCGCCGGCGTCGCCCGCCACCCGCTGCCGGGCGTGTCCGCCGAGAACTGCACCGAGCAGCTGGGCGTCCCGGGCTCCTGGTCCGAGCGCCTGGCCCACTTCCGCATGGCCTTCACCCCGTCCTCCGGCGAGGAGCTGCAGTCCGAGTTCTTCGTCCGCCGCGAGCACGCCGTGGCCGCCATCGGCGAGCTGCGCGCCCTGTCCGACCGCATCACCCCGCTGCTGCTGGTCTCCGAGATCCGCACCGTCGCCGCCGACAAGCTGTGGCTGTCCACCGCCTACGGCCAGGACTCCGTCGGCTTCCACTTCACCTGGAAGCAGCGCCAGGACGAGGTCGAGAAGGTCCTGCCGGTCATGGAGGAGGCCCTGGCCCCGTTCAACGCCCGCCCGCACTGGGGCAAGCTGTTCCACGCCGGCGCCGACGCCGTCGCCGAGCTGTACCCGCGCTTCTCCGACTTCAAGGACCTGGCCGAGCGCATGGACCCCGAGCAGAAGTTCCGCAACGAGTTCCTGGCCCGCAAGGTGTTCGGCAACTGATGAGGATCCG.

Example 14 Bleaching Performance of SOx in Combination with Perhydrolasein HDL Laundry Wash Conditions

The effect of a cleaning composition containing a POx enzyme and aperhydrolase in bleaching a stained fabric is tested.

The effect of bleaching a stained fabric with a composition comprisingperhydrolase M. smegmatis perhydrolase, variant S54V described inPCT/US05/056782 in combination with SOx is tested on fabric stained withtea, or wine or blueberry essentially as described in Example 5 butwithout TAED. The bleaching effect of SOx alone on the stained fabricswatches is performed as described in Example 5. To determine thebleaching effect of a combination of SOx and perhydrolase on fabricswatches stained in the same manner as those tested for SOx alone, 1 ppmof perhydrolase and 5 mM perhydrolase substrate (ester) are combinedwith the sorbitol oxidase and added to the stained swatch disks in themicroplate. The percent soil release is determined and the bleachingperformance of SOx in combination with perhydrolase is calculated.

The bleaching effect of the combination of perhydrolase with SOx issignificantly greater than the bleaching effect obtained with SOx alone.

Therefore, this example illustrates the usefulness of combining a POxand a perhydrolase in cleaning compositions.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

Those of skill in the art readily appreciate that the present inventionis well adapted to carry out the objects and obtain the ends andadvantages mentioned, as well as those inherent therein. Thecompositions and methods described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It is readily apparent to oneskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention. Thus, itshould be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

1. An isolated chimeric polynucleotide comprising a sequence encoding amature polyol oxidase protein (POx), said sequence being operably linkedto a sequence encoding a secretory signal peptide, said signal peptidebeing derived from a prokaryotic microorganism.
 2. The isolatedpolynucleotide of claim 1, wherein said sequence encoding a mature POxis derived from a Streptomyces sp., an Acidothermus sp. or anArthrobacter sp.
 3. The chimeric polynucleotide of claim 1, wherein saidchimeric polynucleotide encodes a polypeptide having a sequence selectedform SEQ ID NO:16, 28, 35, 37, 38, and
 40. 4. The chimericpolynucleotide of claim 1, wherein said sequence encoding said secretorysignal peptide is selected from the signal sequence encoded by the S.coelicolor gene SCO7637, the signal sequence encoding the S. lividansgene SCO 0624 and the signal sequence encoding the B. subtilis geneP43379.
 5. A recombinant expression vector comprising the isolatedchimeric polynucleotide of claim
 2. 6. A host cell comprising therecombinant expression vector of claim
 5. 7. A host cell comprising apolynucleotide encoding a POx polypeptide, wherein said Pox polypeptideis selected from SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 28, 35, 37, 38, and
 40. 8. The host cell of claim 7, wherein saidPOx has sorbitol oxidase and/or xylitol oxidase activity.
 9. The hostcell of claim 7, wherein said polynucleotide is present in the genome ofsaid host cell or in a vector that autonomously replicates in said hostcell.
 10. The host cell of claim 7, wherein said host cell is an S.lividans, a B. subtilis or an Acidothermus cellulolyticus host cell. 11.A method for producing a polypeptide having POx activity comprising: (a)transforming a host cell with a recombinant expression vector, saidexpression vector comprising a polynucleotide sequence encoding a POxpolypeptide having a sequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 28, 35, 37, 38, and 40, (b) growing saidhost cell comprising said recombinant expression vector under conditionssuitable for the expression of said POx polypeptide; and (c) recoveringsaid POx polypeptide.
 12. The method of claim 11, wherein said POxactivity is sorbitol and/or xylitol activity.
 13. The method of claim11, wherein said host cell is selected from Bacillus cells orStreptomyces cells.
 14. The method of claim 11, wherein said expressionof said POx is extracellular.
 15. The method of claim 11, wherein saidexpression of said POx is intracellular.
 16. A cleaning compositioncomprising an effective amount of an isolated POx comprising an aminoacid sequence that is at least about 70% identical to a POx having asequence selected from SEQ ID NO:2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and
 14. 17. The cleaning composition of claim 16, wherein said cleaningcomposition is a detergent.
 18. The cleaning composition of claim 17,further comprising a bleach activator.
 19. The cleaning composition ofclaim 16, further comprising at least one additional enzyme.
 20. Thecleaning composition of claim 19, wherein said at least one additionalenzyme is selected from hemicellulases, peroxidases, proteases,cellulases, xylanases, lipases, phospholipases, esterases, cutinases,pectinases, keratinases, reductases, oxidases, oxidoreductases,perhydrolases, phenoloxidases, lipoxygenases, ligninases, pullulanases,tannases, pentosanases, mannanases, β-glucanases, arabinosidases,hyaluronidases, chondroitinases, laccases, and amylasess, or mixturesthereof.
 21. The cleaning composition of claim 19, wherein said at leastone additional enzyme is a perhydrolase.
 22. The cleaning composition ofclaim 19, wherein said at least one additional enzyme is a glucoseoxidase.
 23. The cleaning composition of claim 19, wherein said cleaningcomposition is a bleaching composition.
 24. A method of cleaning,comprising the step of contacting a hard surface and/or an articlecomprising a fabric with the cleaning composition of claim
 16. 25. Themethod of claim 24, further comprising the step of rinsing said surfaceand/or article after contacting said surface or article with saidcleaning composition.
 26. The method of claim 24, wherein said surfaceand/or article comprising a fabric is stained with a substancecontaining at least one polylol.
 27. The method of claim 26, whereinsaid polyol is D-sorbitol, D-xylitol, D-mannitol, D-ribitol,myo-inositol, glycerol, 1,3,-propanediol or 1,2-propanediol.
 28. Themethod of claim 26, wherein said surface and/or said fabric article issoiled with juice, wine and/or tea.